Heterocyclylalkyne derivatives and their use as modulators of mGluR5 receptors

ABSTRACT

This invention relates to compounds of formula I, 
                         
their use as allosteric modulators of mGluR5 receptor activity, pharmaceutical compositions containing the same, and methods of using the same as agents for the treatment and/or prevention of neurological and psychiatric disorders associated with glutamate dysfunction, such as schizophrenia or cognitive decline, dementia or cognitive impairment, or other pathologies that can be related directly or indirectly to glutamate dysfunction.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/097,482, filed on Dec. 29, 2014, which is incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to heterocyclylalkynes and their use asallosteric modulators of mGluR5 receptor activity, pharmaceuticalcompositions comprising such compounds, and methods of treatmenttherewith. Compounds of the invention can be used for the treatmentand/or prevention of neurological and psychiatric disorders associatedwith glutamate dysfunction such as schizophrenia or cognitive decline,dementia or cognitive impairment, or other pathologies that can berelated either directly or indirectly to glutamate dysfunction.

BACKGROUND TO THE INVENTION

Glutamate is the primary excitatory amino acid in the mammalian centralnervous system. Neurotransmission mediated by glutamate has beendemonstrated to be critical in many physiological processes, such assynaptic plasticity, long term potentiation involved in both learningand memory as well as sensory perception (Riedel et al., Behav. BrainRes. (2003), Vol. 140, pp. 1-47, in review). Furthermore, it has beendemonstrated that an imbalance of glutamate neurotransmission plays acritical role in the pathophysiology of various neurological andpsychiatric diseases.

The excitatory neurotransmission of glutamate is mediated through atleast two different classes of receptors: ionotropic glutamate receptorssuch as the N-methyl-D-aspartate receptor (NMDA),α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) orkainate; and the metabotropic glutamate receptors (mGluR). Theionotropic receptors are ligand gated ion channels and are thought to beresponsible for regulating the rapid neuronal transmission between twoneurons. The metabotropic glutamate receptors are G-protein coupledreceptors (GPCRs) which appear to mediate not only synaptictransmission, but also to regulate the extent of neurotransmitterrelease as well as post synaptic receptor activation.

Dysregulation in glutamatergic neurotransmission, for example throughaltered glutamate release or post-synaptic receptor activation, has beendemonstrated in a variety of neurological as well as psychiatricdisorders. Hypofunction of the NMDA receptor has not only beendemonstrated in Alzheimer's patients, but is increasingly accepted asthe putative cause of schizophrenia (Farber et al., Prog. Brain Res.,(1998), Vol. 116, pp. 421-437, Coyle et al., Cell. and Mol. Neurobiol.,(2006), Vol. 26, pp. 365-384). This is supported by clinical studiesshowing that antagonists of the NMDA receptor induce symptomsindistinguishable to those suffered by schizophrenia patients (Javitt etal., Am J. Psychiatry, (1991), Vol. 148, pp. 1301-1308; Meltzer HY,Biol. Psychiatry, (1999), Vol. 46(10), pp. 1321-1327). Therefore,approaches that could potentiate or normalize NMDA receptor signalinghave the potential to treat neurological and psychiatric disorders.mGluR5, a G protein-coupled receptor that is encoded by the GRM5 gene,belongs to a superfamily of currently eight identified Type III GPCRs,which are unique in that the glutamate ligand binds to a largeextracellular amino-terminal protein domain.

This superfamily is further divided into three groups (Groups I, II andIII) based on amino acid homology as well as the intracellular signalingcascades they regulate (Schoepp et al., Neuropharma, (1999), Vol. 38,pp. 1431-1476) and pharmacological profile. mGluR5 belongs to Group Iand is coupled to the phospholipase C signaling cascade which regulatesintracellular calcium mobilization.

In the central nervous system (CNS), mGluR5 has been demonstrated to beexpressed mainly in the cortex, hippocampus, nucleus accumbens and thecaudate-putamen. These brain regions are known to be involved in memoryformation and cognitive function as well as emotional response. mGluR5has been shown to be localized post-synaptically, adjacent to thepost-synaptic density (Lujan et al., Eur. J. Neurosci. (1996), Vol. 8,pp. 1488-1500). A functional interaction between mGluR5 and the NMDAreceptor has also been demonstrated, where activation of mGluR5potentiates the activation state of the NMDA receptor (Mannaioni et al.,NeuroSci., (2001), Vol. 21, pp. 5925-5924, Rosenbrock et al., Eur. J.Pharma., (2010), Vol. 639, pp. 40-46). Furthermore, activation of mGluR5has been demonstrated in pre-clinical, in vivo models to rescuecognitive impairment as well as psychotic disturbance induced by NMDAreceptor antagonists (Chan et al., Psychopharma. (2008), Vol. 198, pp.141-148). Therefore, activation of mGluR5, and thereby potentiation ornormalization of the NMDA receptor signaling, is a potential mechanismfor the treatment of psychiatric and neurological disorders.

Most agonists of mGluR5 bind the orthosteric glutamate binding site.Since the glutamate binding site between the mGluR family members ishighly conserved, it has been challenging to develop selective mGluR5agonists, which have acceptable CNS penetration and demonstrate in vivoactivity.

An alternative approach to achieve selectivity between the mGluR familymembers is to develop compounds, which bind to an allosteric site, whichis not as highly conserved between the family members. These allostericbinding compounds would not interfere with the natural glutamate bindingand signaling, but modulate the receptor activation state. Allostericligands that have agonistic or inverse agonistic activity in the absenceof orthosteric ligands are termed allosteric agonists or antagonists,respectively. Allosteric ligands lacking effect in the absence oforthosteric ligands are termed modulators (negative or positive).

Positive allosteric modulators of mGluR5 have recently been identified(O'Brien et al., Mol. Pharma. (2003), Vol. 64, pp. 731-740, Lindsley etal., J. Med. Chem. (2004), Vol. 47, pp. 5825-5828), where it has beendetermined that these compounds potentiate mGluR5 activity in thepresence of bound glutamate. In the absence of bound glutamate, themGluR5 positive modulators do not demonstrate any intrinsic activity.

Therefore, these compounds potentiate the natural signaling of mGluR5 asopposed to agonists, which activate the receptor in a permanent,unnatural manner. mGluR5 positive allosteric modulators thereforerepresent an approach to potentiate mGluR5 signaling which in turnpotentiates and normalizes the NMDA receptor hypofunction detected inneurological and psychiatric disorders. mGluR5 negative allostericmodulators are useful to depress the mGluR5 signaling which in turndecreases and normalizes the NMDA receptor hyperfunction detected insome neurological, psychiatric disorders and in more general CNSdisorders. Both types of allosteric modulator can also be related tosome rare disease e.g. without any kind of limitation, Fragile-Xsyndrome, Rett syndrome, Phelan-McDermid syndrome or tuberous sclerosis.

SUMMARY OF THE INVENTION

The invention provides a compound having the general formula I:

or an enantiomer, diastereomer, N-oxide, or a pharmaceuticallyacceptable salt thereof, wherein: R₁ is an alkyl group, an optionallysubstituted mono-, bi- or tricyclic C₁-C₁₃ heterocyclic group containingfrom 1 to 5 heteroatoms selected from N, O, and S, an optionallysubstituted mono-, bi- or tricyclic C₆-C₁₄ aryl group, an optionallysubstituted C₃-C₆ cycloalkyl group, an optionally substituted C₃-C₆cycloalkenyl group, a bond, or an optionally substituted CO, CS, CH, CH₂or SO₂ group;R₂ is absent, or is an optionally substituted mono- or bicyclic C₁-C₉heterocyclic group containing from 1 to 3 heteroatoms selected from N,O, and S, an optionally substituted mono-, bi- or tricyclic C₆-C₁₄ arylgroup, or an optionally substituted group selected from alkyl,cycloalkyl, alkoxy, cycloalkyloxy, aryloxy, cycloalkyl, cycloalkyloxy,heteroaryloxy, alkylthio, amino, N-alkylamino, N,N-dialkylamino,N-alkyl-N-alkoxyamino or N-alkyl-Nalkyloxyamino; R₃ is an optionallysubstituted alkyl group, an optionally substituted mono-, bi- ortricyclic C₁-C₁₃ heterocyclic group containing from 1 to 5 heteroatomsselected from N, O, and S, an optionally substituted mono-, bi- ortricyclic C₆-C₁₄ aryl group, an optionally substituted C₃-C₆ cycloalkylgroup, or an optionally substituted C₃-C₆ cycloalkenyl group; andn is 1-3.

The optional substituents are independently selected from halogen atomsand C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxy, mercapto, nitro, cyano, oxo,halo(C₁-C₆)alkyl, halo(C₁-C₆)alkoxy, C₁-C₆ alkylthio, C₁-C₆alkylsulphonyl, C₁-C₆ alkylcarbonyl, sulphamoyl, C₁-C₆ alkylsulphamoyl,di(C₁-C₆)alkylsulphamoyl, (C₁-C₆)alkoxycarbonyl and(C₁-C₆)alkylcarbonyl(C₁-C₆)alkyl groups, and from groups of the formulae—NR*R*, —C(═O)—NR*R*, -A, —O-A, —C(═O)-A, —(CH₂)q-A, —NR**-A,—C(═O)—NR**-A, —NR**C(═O)-A and —O—C(═O)-A wherein each R* independentlyrepresents a hydrogen atom or a C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆alkylcarbonyl, phenyl or benzyl group, R** represents a hydrogen atom ora C₁-C₆ alkyl group, q is an integer from 1 to 6 and A represents aphenyl group or a C₁-C₈ heterocyclic group containing from 1 to 3heteroatoms selected from N, O and S; a C₁-C₆ cycloalkyl group; eachgroup A being optionally substituted with from 1 to 3 groupsindependently selected from halo, hydroxy, cyano, nitro and C₁-C₆ alkyl,preferably wherein the optional substituents are selected from thegroups consisting of halogen atoms and C₁-C₆ alkyl groups.

The most preferred compounds according to the invention are those inwhich R₁ represents a CO group and n is 1.

In an embodiment of the invention, R₂ preferably is an optionallysubstituted mono- or bicyclic C₁-C₉ heterocyclic group containing from 1to 3 heteroatoms selected from nitrogen, oxygen and sulfur, or anoptionally substituted group chosen from cycloalkyl, alkoxy,cycloalkyloxy, aryloxy, heteroaryloxy, amino, N-alkylamino,N,N-dialkylamino, or N-alkyl-N-alkoxyamino.

For example, when R₂ represents an optionally substituted mono- orbicyclic C₁-C₉ heterocyclic group containing from 1 to 3 heteroatomsselected from nitrogen, oxygen and sulfur, R₂ is preferably a 2-furyl,5-methyl-2-furyl, 3-furyl, 4-morpholinyl, 4-oxanyl, piperidinyl,1-methyl-4-piperidinyl, 4-methylpiperazinyl, 3-(1,5-dimethyl)pyrazolyl,3-pyridylamino, pyrrolidinyl or 4-thiazolyl group.

For example, when R₂ represents an optionally substituted cycloalkylgroup, R₂ is preferably a cyclopentyl or 4-(1,1-difluorocyclohexyl)group.

For example, when R₂ represents an optionally substituted alkoxy group,R₂ is preferably an ethoxy, isopropoxy, 2,2-dimethylpropoxy, t-butoxy or3-methylbutoxy group.

For example, when R₂ represents an optionally substituted cycloalkyloxygroup, R₂ is preferably a cyclopropylmethoxy or cyclopentoxy group.

For example, when R₂ represents an optionally substituted heteroaryloxygroup, R₂ is preferably a 4-oxanyloxy group.

For example, when R₂ represents an optionally substituted amino group,R₂ is preferably an isopropylamino, 2,2-dimethylpropylamino,t-butylamino, 3-pentylamino cyclopentylamino or a 3-pyridylamino group.

For example, when R₂ represents an optionally substituted N-alkylamino,N,N-dialkylamino, or N-alkyl-N-alkoxyamino group, R₂ is preferably anN,N-dimethyl, N,N-diethyl, N-ethyl-N-isopropyl, N-methoxy-N-methyl orN-(2-methoxyethyl)-N-methyl group.

In an alternative embodiment of the invention, R₂ preferably representsa group having the formula:—OR₄wherein R₄ is a C₁-C₁₀ linear or branched alkyl group, a C₁-C₁₀cycloalkyl group or a C₁-C₁₀ heterocyclic group containing at least oneheteroatom selected from N or O.

For example, when R₄ is a C₁-C₁₀ linear or branched alkyl group, R₄ ispreferably an ethyl, isopropyl, 2,2-dimethylpropyl, t-butyl or3-methylbutyl group so that R₂ is preferably an ethoxy, isopropoxy,2,2-dimethylpropoxy, t-butoxy or 3-methylbutoxy group.

For example, when R₄ is a C₁-C₁₀ a cycloalkyl group, R₄ is preferably acyclopropylmethyl or a cyclopentyl group so that R₂ is preferably acyclopropylmethoxy or cyclopentoxy group.

For example, when R₄ is a C₁-C₁₀ heterocyclic group containing at leastone heteroatom selected from N or O, R₄ is preferably a 4-oxanyl groupso that R₂ is a 4-oxanyloxy group.

In an alternative embodiment of the invention, R₂ preferably is asaturated or unsaturated, optionally substituted, five or six memberedhomocyclic group or heterocyclic group containing at least oneheteroatom selected from N or O.

For example, when R₂ is an optionally substituted, five memberedhomocyclic group, R₂ is preferably a cyclopentyl group. When R₂ anoptionally substituted six membered homocyclic group, R₂ is preferably a4-(1,1-difluorocyclohexyl) group.

For example, when R₂ is an optionally substituted, five memberedheterocyclic group containing at least one heteroatom selected from N orO, R₂ is preferably a 2-furyl, 5-methyl-2-furyl, 3-furyl,3-(1,5-dimethyl)pyrazolyl, pyrrolidinyl or 4-thiazolyl group. When R₂ isan optionally substituted, five membered heterocyclic group containingat least one heteroatom selected from N or O, R₂ is preferably a4-morpholinyl, 4-oxanyl, piperidinyl, 1-methyl-4-piperidinyl,4-methylpiperazinylor 3-pyridylamino group.

In an alternative embodiment of the invention, R₂ preferably representsa group having the formula:—NR₅R₆wherein R₅ is a C₁-C₁₀ linear or branched alkyl or alkoxy group orhydrogen; R₆ is a C₁-C₁₀ linear or branched alkyl or alkoxy group, R₅and R₆ being the same or different; or wherein R₅ and R₆ together withthe nitrogen atom form a five or six membered heterocyclic ring.

For example, when R₅ or R₆ is a C₁-C₁₀ linear or branched alkyl group,R₅ or R₆ is preferably a methyl, ethyl, isopropyl, 2,2-dimethylpropyl,t-butyl or 3-pentyl group and R₂ is preferably an N,N-dimethyl,N,N-diethyl, N-ethyl-N-isopropyl, isopropylamino, t-butylamino,3-pentylamino or 2,2-dimethylpropylamino group.

For example, when R₅ or R₆ is a C₁-C₁₀ linear or branched alkoxy group,R₅ or R₆ is preferably a methoxy or 2-methoxyethyl group and R₂ ispreferably an N-methoxy-N-methyl or N-(2-methoxyethyl)-N-methyl group.

For example, when R₅ and R₆ together with the nitrogen atom form a fiveor six membered heterocyclic ring, R₂ is preferably a4-methylpiperazinyl, 4-morpholinyl, piperidinyl or pyrrolidinyl group.

In an embodiment of the invention, R₃ preferably is an optionallysubstituted mono-, bi- or tricyclic C₆-C₁₄ aryl group, an optionallysubstituted, five or six membered heterocyclic group containing at leastone heteroatom selected from N or O, an optionally substituted C₃-C₆cycloalkyl group, or an optionally substituted C₃-C₆ cycloalkenyl group.For example, when R₃ is an optionally substituted mono-, bi- ortricyclic C₆-C₁₄ aryl group, R₃ is preferably a phenyl group. When R₃ isan optionally substituted, five or six membered heterocyclic groupcontaining at least one heteroatom selected from N or O, R₃ ispreferably a pyridyl group.

In an alternative embodiment of the invention, R₃ is most preferably anoptionally substituted phenyl or pyridyl group, with said optionalsubstituents being selected from a C₁-C₁₀ alkyl group or a halide group.For example, R₃ represents a phenyl, 3-methylphenyl, 3-bromophenyl,3-chlorophenyl, 3-fluorophenyl or 6-methyl-2-pyridyl group.

In an alternative embodiment of the invention, R₁ represents anoptionally substituted CO group, R₂ is absent, R₃ represents a phenyl,3-bromophenyl, 3-chlorophenyl, 3-fluorophenyl, 3-methylphenyl or a6-methyl-2-pyridyl group and n is 1.

In an embodiment of the invention, a compound, or an enantiomer,diastereomer, N-oxide, or a pharmaceutically acceptable salt, isprovided according to general formula I selected from:

In a further embodiment of the invention, a pharmaceutical compositionis preferably provided comprising a compound of Formula I,

or an enantiomer, diastereomer, N-oxide, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier,wherein R₁ is an alkyl group, an optionally substituted mono-, bi- ortricyclic C₁-C₁₃ heterocyclic group containing 1 to 5 heteroatomsselected from N, O, and S; an optionally substituted mono-, bi- ortricyclic C₆-C₁₄ aryl group, an optionally substituted C₃-C₆ cycloalkylgroup, or an optionally substituted C₃-C₆ cycloalkenyl group; or a bond,CO, CS, CH, CH₂, SO₂ group optionally substituted by one or more R₂group or substituent; R₂ is absent or is an optionally substituted mono-or bicyclic C₁-C₉ heterocyclic group containing from 1 to 3 heteroatomsselected from nitrogen, oxygen and sulfur, an optionally substitutedmono-, bi- or tricyclic C₆-C₁₄ aryl group, or an optionally substitutedgroup chosen from alkyl, cycloalkyl, alkoxy, cycloalkyloxy, aryloxy,heteroaryloxy, alkylthio, amino, N-alkylamino, N,N-dialkylamino orN-alkyl-N-alkoxyamino; R₃ is an optionally substituted alkyl group, anoptionally substituted mono-, bi- or tricyclic C₁-C₁₃ heterocyclic groupcontaining 1 to 5 heteroatoms selected from N, O, and S; an optionallysubstituted mono-, bi- or tricyclic C₆-C₁₄ aryl group, an optionallysubstituted C₃-C₆ cycloalkyl group, or an optionally substituted C₃-C₆cycloalkenyl group; andn is 1-3.

In another embodiment of the invention, a compound of Formula I,

or an enantiomer, diastereomer, N-oxide, or a pharmaceuticallyacceptable salt thereof, for use in the treatment and/or prevention of aneurological disorder, psychotic disorder, or a psychiatric disorderassociated with glutamate dysfunction is preferably provided, wherein R₁is an alkyl group, an optionally substituted mono-, bi- or tricyclicC₁-C₁₃ heterocyclic group containing 1 to 5 heteroatoms selected from N,O, and S; an optionally substituted mono-, bi- or tricyclic C₆-C₁₄ arylgroup, an optionally substituted C₃-C₆ cycloalkyl group, or anoptionally substituted C₃-C₆ cycloalkenyl group; or a bond, CO, CS, CH,CH₂, SO₂ group optionally substituted by one or more R₂ group orsubstituent; R₂ is absent or is an optionally substituted mono- orbicyclic C₁-C₉ heterocyclic group containing from 1 to 3 heteroatomsselected from nitrogen, oxygen and sulfur, an optionally substitutedmono-, bi- or tricyclic C₆-C₁₄ aryl group, or an optionally substitutedgroup chosen from alkyl, cycloalkyl, alkoxy, cycloalkyloxy, aryloxy,heteroaryloxy, alkylthio, amino, N-alkylamino, N,N-dialkylamino orN-alkyl-N-alkoxyamino; R₃ is an optionally substituted alkyl group, anoptionally substituted mono-, bi- or tricyclic C₁-C₁₃ heterocyclic groupcontaining 1 to 5 heteroatoms selected from N, O, and S; an optionallysubstituted mono-, bi- or tricyclic C₆-C₁₄ aryl group, an optionallysubstituted C₃-C₆ cycloalkyl group, or an optionally substituted C₃-C₆cycloalkenyl group; and n is 1-3.

In an embodiment of the invention, a compound according to Formula I isused in the treatment and/or prevention of a neurological disorder,psychotic disorder, or a psychiatric disorder associated with glutamatedysfunction.

Preferably the neurological disorder, psychotic disorder, or psychiatricdisorder associated with glutamate dysfunction is schizophrenia,schizoaffective disorder, substance induced psychotic disorder,age-associated learning and memory impairments or losses, post strokedementia, deficits in concentration, mild cognitive impairment,cognitive dysfunction in Alzheimers disease, cognitive dysfunction ofschizophrenia, cognitive decline, dementia or cognitive impairment.

More preferably the disorder is Fragile-X syndrome, Rett syndrome,Phelan-McDermid syndrome, or tuberous sclerosis.

TERMS AND DEFINITIONS USED

Except where stated otherwise, the following definitions applythroughout the present specification and claims. These definitions applyregardless of whether a term is used by itself or in combination withother terms. For example, the definition of “alkyl” applies not only toalkyl groups per se, but also to the alkyl portions of alkoxy,alkylamino, alkylthio or alkylcarbonyl groups etc. Furthermore allranges described for a chemical group, for example “from 1 to 13 carbonatoms” or “C₁-C₆ alkyl” include all combinations and sub-combinations ofranges and specific numbers of carbon atoms therein.

“Alkyl” means a straight chain or branched chain aliphatic hydrocarbongroup having from 1 to 20 carbon atoms in the chain. Preferred alkylgroups have from 1 to 12 carbon atoms in the chain. More preferred alkylgroups have from 1 to 6 carbon atoms in the chain. “Lower alkyl” meansan alkyl group having about 1 to about 6 carbon atoms in the chain whichmay be straight or branched. Examples of suitable alkyl groups includemethyl, ethyl, n-propyl, isopropyl, sec-butyl, n-butyl, and t-butyl.

“Alkenyl” means a straight chain or branched chain aliphatic hydrocarbongroup having at least one carbon-carbon double bond and having from 2 to15 carbon atoms in the chain. Preferred alkenyl groups have from 2 to 12carbon atoms in the chain. More preferred alkenyl groups have from 2 to6 carbon atoms in the chain. “Lower alkenyl” means an alkenyl grouphaving 2 to about 6 carbon atoms in the chain, which may be straight orbranched. Examples of suitable alkenyl groups include ethenyl, propenyl,isopropenyl, n-butenyl, 1-hexenyl and 3-methylbut-2-enyl.

“Alkynyl” means a straight chain or branched chain aliphatic hydrocarbongroup having at least one carbon-carbon triple bond and having from 2 to15 carbon atoms in the chain. Preferred alkynyl groups have from 2 to 12carbon atoms in the chain. More preferred alkynyl groups have from 2 to6 carbon atoms in the chain. “Lower alkynyl” means an alkynyl grouphaving 2 to about 6 carbon atoms in the chain, which may be straight orbranched. Examples of suitable alkynyl groups include ethynyl, propynyland 2-butynyl.

“Mono-, bi-, or tricyclic heterocyclic” means an aromatic ornon-aromatic saturated mono- bi- or tricyclic ring system having from 2to 14 ring carbon atoms, and containing from 1 to 5 ring atoms selectedfrom N, O and S, alone or in combination. Bi- and tricyclic heterocyclicgroups are fused at 2 or 4 points or joined at one point via a bond or aheteroatom linker (O, S, NH, or N(C₁-C₆ alkyl). The “mono- bi- ortricyclic heterocyclic” can be optionally substituted on the ring byreplacing an available hydrogen on the ring by one or more substituentswhich may be the same or different. The nitrogen or sulphur atom of theheterocyclic can be optionally oxidized to the corresponding N-oxide,S-oxide or S-dioxide. Examples of suitable heterocyclics includefuranyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl,pyridyl, pyrimidyl, pyridazinyl, thiazolyl, triazolyl, tetrazolyl,thienyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl,indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl andbenzoisoxazolyl, aziridinyl, piperidinyl, pyrrolidinyl, piperazinyl,tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiophenyl, morpholinyland thiomorpholinyl.

Heterocyclics with aromatic characteristics may be referred to asheteroaryls or heteroaromatics. Examples of suitable heteroaromaticsinclude furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl,pyrrolyl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, triazolyl,tetrazolyl, thienyl, carbazolyl, benzimidazolyl, benzothienyl,benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl,benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl,benzoisoxazolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,3-phenylpyridine, 3-cyclohexylpyridine, 3-(pyridin-3-yl) morpholine,3-phenylisoxazole and 2-(piperidin-1-yl)pyrimidine.

“Mono-, bi- or tricyclic aryl” means an aromatic monocyclic, bicyclic ortricyclic ring system comprising 6 to 14 carbon atoms. Bi- and tricyclicaryl groups are fused at 2 or 4 points or joined at one point via a bondor a heteroatom linker (O, S, NH, or N(C₁-C₆ alkyl) (e.g., biphenyl,1-phenylnapthyl). The aryl group can be optionally substituted on thering with one or more substituents, preferably 1 to 6 substituents,which may be the same or different. Examples of suitable aryl groupsinclude phenyl and naphthyl.

“Cycloalkyl” means a monocyclic or bicyclic carbon ring system havingfrom 3 to 14 carbon atoms, preferably from 3 to 6 carbon atoms. Thecycloalkyl can be optionally substituted on the ring by replacing anavailable hydrogen on the ring by one or more substituents which may bethe same or different. Examples of suitable monocyclic cycloalkylsinclude cyclopropyl, cyclopentyl, cyclohexyl and cycloheptyl. Examplesof suitable multicyclic cycloalkyls include 1-decalinyl, norbornyl andadamantyl.

“Cycloalkenyl” has a meaning corresponding to that of cycloalkyl, butwith one or two double bonds within the ring (e.g., cyclohexenyl,cyclohexadiene).

“Amines” are derivatives of ammonia, wherein one or more hydrogen atomshave been replaced by a substituent such as an alkyl or aryl group.These may respectively be called alkylamines and arylamines; amines inwhich both types of substituent are attached to one nitrogen atom may becalled alkylarylamines.

Amines can be further organized into four sub-categories. Primary aminesarise when one of the three hydrogen atoms in ammonia is replaced by analkyl or aromatic group (an N-alkylamino or N-arylamino respectively).Examples of suitable primary alkyl amines include methylamine orethanolamine, or aniline (phenylamine) as an example of an aromaticamine. Secondary amines have two organic substituents (independentlyalkyl or aryl groups) bound to the nitrogen atom together with onehydrogen (or no hydrogen if one of the substituent bonds is double).Examples of suitable secondary amines include dimethylamine andmethylethanolamine, while an example of an aromatic amine would bediphenylamine. Such compounds may also be referred to as“N,N-dialkylamino”, “N,N-diarylamino” or “N,N-alkylarylamino” groupsdepending on the nature of the substituents. A secondary aminesubstituted by an alkoxy group, as defined herein, would be termed an“N-alkyl-N-alkoxyamino” compound for example. In tertiary amines, allthree hydrogen atoms are replaced by organic substituents, such astrimethylamine. The final sub-category is cyclic amines which are eithersecondary or tertiary amines. Examples of suitable cyclic amines includethe 3-member ring aziridine and the six-membered ring piperidine.N-methylpiperidine and N-phenylpiperidine are suitable examples ofcyclic tertiary amines.

“Amides” are compounds with a nitrogen atom attached to a carbonylgroup, thus having the structure R—CO—NR′R″, with groups R′ and R″ beingindependently selected from alkyl or aromatic groups as defined herein.For example when R′ is hydrogen and R″ is a 3-pyridyl group, theresulting amide has a 3-pyridylamino substituent. Alternatively when R′is hydrogen and R″ is a cyclopentyl group, the resulting amide has acyclopentylamino substituent.

“Halogen” or “Hal” means fluorine, chlorine, bromine or iodine.Preferred halogens are fluorine, chlorine or bromine, and most preferredare fluorine and chlorine.

The term “acyl”, whether used alone, or within a term such as“acylamino”, denotes a radical provided by the residue after removal ofhydroxyl from an organic acid. The term “acylamino” refers an aminoradical substituted with an acyl group. An example of an “acylamino”radical is CH₃C(═O)—NH— where the amine may be further substituted withalkyl, aryl or aralkyl.

An asterisk may be used in sub-formulas to indicate the bond, which isconnected to a parent or core molecule as defined herein.

Stereochemistry

Unless specifically indicated, throughout the specification and claims,a given chemical formula or name shall encompass tautomers and allstereo, optical and geometrical isomers (e.g. enantiomers,diastereomers, E/Z isomers etc.) and racemates thereof. This includesmixtures in different proportions of the separate enantiomers, mixturesof diastereomers, or mixtures of any of the foregoing forms where suchisomers and enantiomers exist, as well as salts, includingpharmaceutically acceptable salts and solvates thereof such as hydrates,solvates of the free compounds or solvates of a salt of the compound.

Derivatives of Compounds of the Invention

The invention further encompasses salts, solvates, hydrates, N-oxides,produgs and active metabolites of the compounds of formula I.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication, andcommensurate with a reasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salts” refers toderivatives of the disclosed compounds wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. For example, such salts include salts from ammonia, L-arginine,betaine, benethamine, benzathine, calcium hydroxide, choline, deanol,diethanolamine (2,2′-iminobis(ethanol)), diethylamine,2-(diethylamino)-ethanol, 2-aminoethanol, ethylenediamine,N-ethyl-glucamine, hydrabamine, 1H-imidazole, lysine, magnesiumhydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassiumhydroxide, 1-(2-hydroxyethyl)-pyrrolidine, sodium hydroxide,triethanolamine (2,2′,2″-nitrilotris(ethanol)), tromethamine, zinchydroxide, acetic acid, 2,2-dichloro-acetic acid, adipic acid, alginicacid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoicacid, 2,5-dihydroxybenzoic acid, 4-acetamido-benzoic acid, (+)-camphoricacid, (+)-camphor-10-sulfonic acid, carbonic acid, cinnamic acid, citricacid, cyclamic acid, decanoic acid, dodecylsulfuric acid,ethane-1,2-disulfonic acid, ethanesulfonic acid,2-hydroxy-ethanesulfonic acid, ethylenediaminetetraacetic acid, formicacid, fumaric acid, galactaric acid, gentisic acid, D-glucoheptonicacid, D-gluconic acid, D-glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycine, glycolic acid,hexanoic acid, hippuric acid, hydrobromic acid, hydrochloric acid,isobutyric acid, DL-lactic acid, lactobionic acid, lauric acid, lysine,maleic acid, (−)-L-malic acid, malonic acid, DL-mandelic acid,methanesulfonic acid, galactaric acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,nitric acid, octanoic acid, oleic acid, orotic acid, oxalic acid,palmitic acid, pamoic acid (embonic acid), phosphoric acid, propionicacid, (−)-L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid,sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid,(+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid andundecylenic acid. Further pharmaceutically acceptable salts can beformed with cations from metals such as aluminium, calcium, lithium,magnesium, potassium, sodium, zinc and the like (see Pharmaceuticalsalts, Berge, S. M. et al., J. Pharm. Sci., (1977), Vol. 66, pp. 1-19).

Pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound, which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha sufficient amount of the appropriate base or acid in water or in anorganic diluent like ether, ethyl acetate, ethanol, isopropanol, oracetonitrile, or a mixture thereof.

Salts of other acids than those mentioned above which for example areuseful for purifying or isolating the compounds of the present invention(e.g. trifluoro acetate salts), also comprise a part of the invention.

Typically, a pharmaceutically acceptable salt of a compound of formula Imay be readily prepared by using a desired acid or base as appropriate.The salt may precipitate from solution and be collected by filtration ormay be recovered by evaporation of the solvent. For example, an aqueoussolution of an acid such as hydrochloric acid may be added to an aqueoussuspension of a compound of formula I and the resulting mixtureevaporated to dryness (lyophilized) to obtain the acid addition salt asa solid. Alternatively, a compound of formula I may be dissolved in asuitable solvent, for example an alcohol such as isopropanol, and theacid may be added in the same solvent or another suitable solvent. Theresulting acid addition salt may then be precipitated directly, or byaddition of a less polar solvent such as diisopropyl ether or hexane,and isolated by filtration.

The acid addition salts of the compounds of formula I may be prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. The free base formmay be regenerated by contacting the salt form with a base and isolatingthe free base in the conventional manner. The free base forms differfrom their respective salt forms somewhat in certain physical propertiessuch as solubility in polar solvents, but otherwise the salts areequivalent to their respective free base for purposes of the invention.

Also included are both total and partial salts, that is to say saltswith 1, 2 or 3, preferably 2, equivalents of base per mole of acid offormula I or salts with 1, 2 or 3 equivalents, preferably 1 equivalent,of acid per mole of base of formula I.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.

The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid.

Compounds of the invention may have both a basic and an acidic centerand may therefore be in the form of zwitterions or internal salts.

Typically, a pharmaceutically acceptable salt of a compound of formula Imay be readily prepared by using a desired acid or base as appropriate.The salt may precipitate from solution and be collected by filtration ormay be recovered by evaporation of the solvent. For example, an aqueoussolution of an acid such as hydrochloric acid may be added to an aqueoussuspension of a compound of formula I and the resulting mixtureevaporated to dryness (lyophilized) to obtain the acid addition salt asa solid. Alternatively, a compound of formula I may be dissolved in asuitable solvent, for example an alcohol such as isopropanol, and theacid may be added in the same solvent or another suitable solvent. Theresulting acid addition salt may then be precipitated directly, or byaddition of a less polar solvent such as diisopropyl ether or hexane,and isolated by filtration.

Those skilled in the art of organic chemistry will appreciate that manyorganic compounds can form complexes with solvents in which they arereacted or from which they are precipitated or crystallized. Thesecomplexes are known as “solvates”. For example, a complex with water isknown as a “hydrate”. Solvates of the compound of the invention arewithin the scope of the invention. The salts of the compound of formulaI may form solvates (e.g., hydrates) and the invention also includes allsuch solvates. The meaning of the word “solvates” is well known to thoseskilled in the art as a compound formed by interaction of a solvent anda solute (i.e., solvation). Techniques for the preparation of solvatesare well established in the art (see, for example, Brittain.Polymorphism in Pharmaceutical Solids. Marcel Decker, New York, 1999.).

The invention also encompasses N-oxides of the compounds of formulas I.The term “N-oxide” means that for heterocycles containing an otherwiseunsubstituted sp² N atom, the N atom may bear a covalently bound O atom,i.e., —N→O. Examples of such N-oxide substituted heterocycles includepyridyl N-oxides, pyrimidyl N-oxides, pyrazinyl N-oxides and pyrazolylN-oxides.

The invention also encompasses prodrugs of the compounds of formula I,i.e., compounds which release an active parent drug according to formulaI in vivo when administered to a mammalian subject. A prodrug is apharmacologically active or more typically an inactive compound that isconverted into a pharmacologically active agent by a metabolictransformation. Prodrugs of a compound of formula I are prepared bymodifying functional groups present in the compound of formula I in sucha way that the modifications may be cleaved in vivo to release theparent compound. In vivo, a prodrug readily undergoes chemical changesunder physiological conditions (e.g., are acted on by naturallyoccurring enzyme(s)) resulting in liberation of the pharmacologicallyactive agent. Prodrugs include compounds of formula I wherein a hydroxy,amino, or carboxy group of a formula I compound is bonded to any groupthat may be cleaved in vivo to regenerate the free hydroxyl, amino orcarboxy group, respectively. Examples of prodrugs include esters (e.g.,acetate, formate, and benzoate derivatives) of compounds of formula I orany other derivative, which upon being brought to the physiological pHor through enzyme action is converted to the active parent drug.Conventional procedures for the selection and preparation of suitableprodrug derivatives are described in the art (see, for example,Bundgaard. Design of Prodrugs. Elsevier, 1985).

Prodrugs may be administered in the same manner as the active ingredientto which they convert or they may be delivered in a reservoir form,e.g., a transdermal patch or other reservoir which is adapted to permit(by provision of an enzyme or other appropriate reagent) conversion of aprodrug to the active ingredient slowly over time, and delivery of theactive ingredient to the patient.

The invention also encompasses metabolites. A “metabolite” of a compounddisclosed herein is a derivative of a compound which is formed when thecompound is metabolised. The term “active metabolite” refers to abiologically active derivative of a compound, which is formed when thecompound is metabolised. The term “metabolised” refers to the sum of theprocesses by which a particular substance is changed in the living body.In brief, all compounds present in the body are manipulated by enzymeswithin the body in order to derive energy and/or to remove them from thebody. Specific enzymes produce specific structural alterations to thecompound. For example, cytochrome P450 catalyses a variety of oxidativeand reductive reactions while uridine diphosphate glucuronyltransferasescatalyse the transfer of an activated glucuronic-acid molecule toaromatic alcohols, aliphatic alcohols, carboxylic acids, amines and freesulphydryl groups. Further information on metabolism may be obtainedfrom The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill(1996), pages 11-17.

Metabolites of the compounds disclosed herein can be identified eitherby administration of compounds to a host and analysis of tissue samplesfrom the host, or by incubation of compounds with hepatic cells in vitroand analysis of the resulting compounds. Both methods are well known inthe art.

The term “carrier” refers to a diluent, excipient, and/or vehicle withwhich an active compound is administered. The pharmaceuticalcompositions of the invention may contain combinations of more than onecarrier. Such pharmaceutical carriers can be sterile liquids, such aswater, saline solutions, aqueous dextrose solutions, aqueous glycerolsolutions, and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or aqueous solution saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin,18th Edition.

A “pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes an excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the present application includes both one and more than one suchexcipient.

The compounds of the invention may be formulated for administration inany convenient way for use in human or veterinary medicine and theinvention therefore includes within its scope pharmaceuticalcompositions comprising a compound of the invention adapted for use inhuman or veterinary medicine. Such compositions may be presented for usein a conventional manner with the aid of one or more suitable carriers.Acceptable carriers for therapeutic use are well-known in thepharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier can be selected with regard to theintended route of administration and standard pharmaceutical practice.The pharmaceutical compositions may comprise as, in addition to, thecarrier any suitable binder(s), lubricant(s), suspending agent(s),coating agent(s), and/or solubilizing agent(s).

Pharmaceutical Compositions Comprising a Compound of Formula I

While it is possible that a compound I may be administered as the bulksubstance, it is preferable to present the active ingredient in apharmaceutical formulation, e.g., wherein the agent is in admixture witha pharmaceutically acceptable carrier selected with regard to theintended route of administration and standard pharmaceutical practice.

Accordingly, the invention further provides a pharmaceutical compositioncomprising a compound of formula I or a solvate, hydrate, enantiomer,diastereomer, N-oxide or pharmaceutically acceptable salt thereof inadmixture with a pharmaceutically acceptable carrier. The term “carrier”refers to a diluent, excipient, and/or vehicle with which an activecompound is administered.

A compound of formula I may be used in combination with other therapiesand/or active agents. Accordingly, the invention provides, in a furtheraspect, a pharmaceutical composition comprising a compound of formula Ior a solvate, hydrate, enantiomer, diastereomer, N-oxide orpharmaceutically acceptable salt thereof, a second active agent, and apharmaceutically acceptable carrier.

The pharmaceutical compositions may comprise as, in addition to, thecarrier any suitable binder, lubricant, suspending agent, coating agentand/or solubilizing agent.

Preservatives, stabilizers, dyes and flavouring agents also may beprovided in the pharmaceutical composition. Antioxidants and suspendingagents may be also used.

The compounds of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the compounds of the invention may beprepared by processes known in the art, for example see WO02/00196.

Routes of Administration and Unit Dosage Forms

The routes for administration include oral (e.g., as a tablet, capsule,or as an ingestible solution), topical, mucosal (e.g., as a nasal sprayor aerosol for inhalation), nasal, parenteral (e.g., by an injectableform), gastrointestinal, intraspinal, intraperitoneal, intramuscular,intravenous, intrauterine, intraocular, intradermal, intracranial,intratracheal, intravaginal, intracerebroventricular, intracerebral,subcutaneous, ophthalmic (including intravitreal or intracameral),transdermal, rectal, buccal, epidural and sublingual. The compositionsof the invention may be especially formulated for any of thoseadministration routes. In preferred embodiments, the pharmaceuticalcompositions of the invention are formulated in a form that is suitablefor oral delivery.

There may be different composition/formulation requirements depending onthe different delivery systems. It is to be understood that not all ofthe compounds need to be administered by the same route. Likewise, ifthe composition comprises more than one active component, then thosecomponents may be administered by different routes. By way of example,the pharmaceutical composition of the invention may be formulated to bedelivered using a mini-pump or by a mucosal route, for example, as anasal spray or aerosol for inhalation or ingestible solution, orparenterally in which the composition is formulated by an injectableform, for delivery, by, for example, an intravenous, intramuscular orsubcutaneous route. Alternatively, the formulation may be designed to bedelivered by multiple routes.

Where the agent is to be delivered mucosally through thegastrointestinal mucosa, it should be able to remain stable duringtransit though the gastrointestinal tract; for example, it should beresistant to proteolytic degradation, stable at acid pH and resistant tothe detergent effects of bile. For example, the compound of Formula Imay be coated with an enteric coating layer. The enteric coating layermaterial may be dispersed or dissolved in either water or in a suitableorganic solvent. As enteric coating layer polymers, one or more,separately or in combination, of the following can be used; e.g.,solutions or dispersions of methacrylic acid copolymers, celluloseacetate phthalate, cellulose acetate butyrate, hydroxypropylmethylcellulose phthalate, hydroxypropyl methylcellulose acetatesuccinate, polyvinyl acetate phthalate, cellulose acetate trimellitate,carboxymethylethylcellulose, shellac or other suitable enteric coatinglayer polymer(s). For environmental reasons, an aqueous coating processmay be preferred. In such aqueous processes methacrylic acid copolymersare most preferred.

When appropriate, the pharmaceutical compositions can be administered byinhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose, or in capsules or ovules either alone or inadmixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or they can beinjected parenterally, for example intravenously, intramuscularly orsubcutaneously. For buccal or sublingual administration the compositionsmay be administered in the form of tablets or lozenges, which can beformulated in a conventional manner.

When the composition of the invention is to be administeredparenterally, such administration includes one or more of:intravenously, intraarterially, intraperitoneally, intrathecally,intraventricularly, intraurethrally, intrasternally, intracranially,intramuscularly or subcutaneously administering the agent; and/or byusing infusion techniques.

Pharmaceutical compositions of the invention can be administeredparenterally, e.g., by infusion or injection. Pharmaceuticalcompositions suitable for injection or infusion may be in the form of asterile aqueous solution, a dispersion or a sterile powder that containsthe active ingredient, adjusted, if necessary, for preparation of such asterile solution or dispersion suitable for infusion or injection. Thispreparation may optionally be encapsulated into liposomes. In all cases,the final preparation must be sterile, liquid, and stable underproduction and storage conditions. To improve storage stability, suchpreparations may also contain a preservative to prevent the growth ofmicroorganisms. Prevention of the action of micro-organisms can beachieved by the addition of various antibacterial and antifungal agents,e.g., paraben, chlorobutanol, or acsorbic acid. In many cases isotonicsubstances are recommended, e.g., sugars, buffers and sodium chloride toassure osmotic pressure similar to those of body fluids, particularlyblood. Prolonged absorption of such injectable mixtures can be achievedby introduction of absorption-delaying agents, such as aluminiummonostearate or gelatin.

Dispersions can be prepared in a liquid carrier or intermediate, such asglycerin, liquid polyethylene glycols, triacetin oils, and mixturesthereof. The liquid carrier or intermediate can be a solvent or liquiddispersive medium that contains, for example, water, ethanol, a polyol(e.g., glycerol, propylene glycol or the like), vegetable oils,non-toxic glycerine esters and suitable mixtures thereof. Suitableflowability may be maintained, by generation of liposomes,administration of a suitable particle size in the case of dispersions,or by the addition of surfactants.

For parenteral administration, the compound is best used in the form ofa sterile aqueous solution, which may contain other substances, forexample, enough salts or glucose to make the solution isotonic withblood. The aqueous solutions should be suitably buffered (preferably toa pH of from 3 to 9), if necessary. The preparation of suitableparenteral formulations under sterile conditions is readily accomplishedby standard pharmaceutical techniques well-known to those skilled in theart.

Sterile injectable solutions can be prepared by mixing a compound offormula I with an appropriate solvent and one or more of theaforementioned carriers, followed by sterile filtering. In the case ofsterile powders suitable for use in the preparation of sterileinjectable solutions, preferable preparation methods include drying invacuum and lyophilization, which provide powdery mixtures of thealdosterone receptor antagonists and desired excipients for subsequentpreparation of sterile solutions.

The compounds according to the invention may be formulated for use inhuman or veterinary medicine by injection (e.g., by intravenous bolusinjection or infusion or via intramuscular, subcutaneous or intrathecalroutes) and may be presented in unit dose form, in ampoules, or otherunit-dose containers, or in multi-dose containers, if necessary with anadded preservative. The compositions for injection may be in the form ofsuspensions, solutions, or emulsions, in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing,solubilizing and/or dispersing agents. Alternatively the activeingredient may be in sterile powder form for reconstitution with asuitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds of the invention can be administered (e.g., orally ortopically) in the form of tablets, capsules, ovules, elixirs, solutionsor suspensions, which may contain flavouring or colouring agents, forimmediate-, delayed-, modified-, sustained-, pulsed- orcontrolled-release applications.

The compounds of the invention may also be presented for human orveterinary use in a form suitable for oral or buccal administration, forexample in the form of solutions, gels, syrups, mouth washes orsuspensions, or a dry powder for constitution with water or othersuitable vehicle before use, optionally with flavouring and colouringagents. Solid compositions such as tablets, capsules, lozenges,pastilles, pills, boluses, powder, pastes, granules, bullets or premixpreparations may also be used. Solid and liquid compositions for oraluse may be prepared according to methods well-known in the art. Suchcompositions may also contain one or more pharmaceutically acceptablecarriers and excipients which may be in solid or liquid form.

The tablets may contain excipients such as microcrystalline cellulose,lactose, sodium citrate, calcium carbonate, dibasic calcium phosphateand glycine, disintegrants such as starch (preferably corn, potato ortapioca starch), sodium starch glycolate, croscarmellose sodium andcertain complex silicates, and granulation binders such aspolyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose (HPC), sucrose, gelatin and acacia.

Additionally, lubricating agents such as magnesium stearate, stearicacid, glyceryl behenate and talc may be included.

The compositions may be administered orally, in the form of rapid orcontrolled release tablets, microparticles, mini tablets, capsules,sachets, and oral solutions or suspensions, or powders for thepreparation thereof. In addition to the new solid-state forms ofpantoprazole of the invention as the active substance, oral preparationsmay optionally include various standard pharmaceutical carriers andexcipients, such as binders, fillers, buffers, lubricants, glidants,dyes, disintegrants, odourants, sweeteners, surfactants, mold releaseagents, antiadhesive agents and coatings. Some excipients may havemultiple roles in the compositions, e.g., act as both binders anddisintegrants.

Examples of pharmaceutically acceptable disintegrants for oralcompositions include starch, pre-gelatinized starch, sodium starchglycolate, sodium carboxymethylcellulose, croscarmellose sodium,microcrystalline cellulose, alginates, resins, surfactants, effervescentcompositions, aqueous aluminum silicates and cross-linkedpolyvinylpyrrolidone.

Examples of pharmaceutically acceptable binders for oral compositionsinclude acacia; cellulose derivatives, such as methylcellulose,carboxymethylcellulose, hydroxypropylmethylcellulose,hydroxypropylcellulose or hydroxyethylcellulose; gelatin, glucose,dextrose, xylitol, polymethacrylates, polyvinylpyrrolidone, sorbitol,starch, pre-gelatinized starch, tragacanth, xanthane resin, alginates,magnesium-aluminum silicate, polyethylene glycol or bentonite.

Examples of pharmaceutically acceptable fillers for oral compositionsinclude lactose, anhydrolactose, lactose monohydrate, sucrose, dextrose,mannitol, sorbitol, starch, cellulose (particularly microcrystallinecellulose), dihydro- or anhydro-calcium phosphate, calcium carbonate andcalcium sulphate.

Examples of pharmaceutically acceptable lubricants useful in thecompositions of the invention include magnesium stearate, talc,polyethylene glycol, polymers of ethylene oxide, sodium lauryl sulphate,magnesium lauryl sulphate, sodium oleate, sodium stearyl fumarate, andcolloidal silicon dioxide.

Examples of suitable pharmaceutically acceptable odourants for the oralcompositions include synthetic aromas and natural aromatic oils such asextracts of oils, flowers, fruits (e.g., banana, apple, sour cherry,peach) and combinations thereof, and similar aromas. Their use dependson many factors, the most important being the organoleptic acceptabilityfor the population that will be taking the pharmaceutical compositions.

Examples of suitable pharmaceutically acceptable dyes for the oralcompositions include synthetic and natural dyes such as titaniumdioxide, beta-carotene and extracts of grapefruit peel.

Examples of useful pharmaceutically acceptable coatings for the oralcompositions, typically used to facilitate swallowing, modify therelease properties, improve the appearance, and/or mask the taste of thecompositions include hydroxypropylmethylcellulose,hydroxypropylcellulose and acrylate-methacrylate copolymers.

Examples of pharmaceutically acceptable sweeteners for the oralcompositions include aspartame, saccharin, saccharin sodium, sodiumcyclamate, xylitol, mannitol, sorbitol, lactose and sucrose.

Examples of pharmaceutically acceptable buffers include citric acid,sodium citrate, sodium bicarbonate, dibasic sodium phosphate, magnesiumoxide, calcium carbonate and magnesium hydroxide.

Examples of pharmaceutically acceptable surfactants include sodiumlauryl sulphate and polysorbates.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the agent may becombined with various sweetening or flavoring agents, coloring matter ordyes, with emulsifying and/or suspending agents and with diluents suchas water, ethanol, propylene glycol and glycerin, and combinationsthereof.

The compounds of the invention may also, for example, be formulated assuppositories e.g., containing conventional suppository bases for use inhuman or veterinary medicine or as pessaries e.g., containingconventional pessary bases.

The compounds according to the invention may be formulated for topicaladministration, for use in human and veterinary medicine, in the form ofointments, creams, gels, hydrogels, lotions, solutions, shampoos,powders (including spray or dusting powders), pessaries, tampons,sprays, dips, aerosols, drops (e.g., eye ear or nose drops) or pour-ons.

For application topically to the skin, the agent of the invention can beformulated as a suitable ointment containing the active compoundsuspended or dissolved in, for example, a mixture with one or more ofthe following: mineral oil, liquid petrolatum, white petrolatum,propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifyingwax, sorbitan monostearate, a polyethylene glycol, liquid paraffin,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol, and water. Such compositions may also contain otherpharmaceutically acceptable excipients, such as polymers, oils, liquidcarriers, surfactants, buffers, preservatives, stabilizers,antioxidants, moisturizers, emollients, colourants, and odourants.

Examples of pharmaceutically acceptable polymers suitable for suchtopical compositions include acrylic polymers; cellulose derivatives,such as carboxymethylcellulose sodium, methylcellulose orhydroxypropylcellulose; natural polymers, such as alginates, tragacanth,pectin, xanthan and cytosan.

Examples of suitable pharmaceutically acceptable oils which are souseful include mineral oils, silicone oils, fatty acids, alcohols, andglycols.

Examples of suitable pharmaceutically acceptable liquid carriers includewater, alcohols or glycols such as ethanol, isopropanol, propyleneglycol, hexylene glycol, glycerol and polyethylene glycol, or mixturesthereof in which the pseudopolymorph is dissolved or dispersed,optionally with the addition of non-toxic anionic, cationic or non-ionicsurfactants, and inorganic or organic buffers.

Examples of pharmaceutically acceptable preservatives include sodiumbenzoate, ascorbic acid, esters of p-hydroxybenzoic acid and variousantibacterial and antifungal agents such as solvents, for exampleethanol, propylene glycol, benzyl alcohol, chlorobutanol, quaternaryammonium salts, and parabens (such as methyl paraben, ethyl paraben andpropyl paraben).

Examples of pharmaceutically acceptable stabilizers and antioxidantsinclude ethylenediaminetetraacetic acid (EDTA), thiourea, tocopherol andbutyl hydroxyanisole.

Examples of pharmaceutically acceptable moisturizers include glycerine,sorbitol, urea and polyethylene glycol.

Examples of pharmaceutically acceptable emollients include mineral oils,isopropyl myristate, and isopropyl palmitate.

The compounds may also be dermally or transdermally administered, forexample, by use of a skin patch.

For ophthalmic use, the compounds can be formulated as micronizedsuspensions in isotonic, pH adjusted, sterile saline, or, preferably, assolutions in isotonic, pH adjusted, sterile saline, optionally incombination with a preservative such as a benzylalkonium chloride.

As indicated, the compounds of the invention can be administeredintranasally or by inhalation and is conveniently delivered in the formof a dry powder inhaler or an aerosol spray presentation from apressurized container, pump, spray or nebulizer with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkanesuch as 1,1,1,2-tetrafluoroethane (HFA 134AT) or1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide or othersuitable gas. In the case of a pressurized aerosol, the dosage unit maybe determined by providing a valve to deliver a metered amount. Thepressurized container, pump, spray or nebulizer may contain a solutionor suspension of the active compound, e.g., using a mixture of ethanoland the propellant as the solvent, which may additionally contain alubricant, e.g., sorbitan trioleate.

Capsules and cartridges (made, for example, from gelatin) for use in aninhaler or insufflator may be formulated to contain a powder mix of thecompound and a suitable powder base such as lactose or starch.

For topical administration by inhalation the compounds according to theinvention may be delivered for use in human or veterinary medicine via anebulizer.

The pharmaceutical compositions of the invention may contain from 0.01to 99% weight per volume of the active material. For topicaladministration, for example, the composition will generally contain from0.01-10%, more preferably 0.01-1% of the active material.

The active agents can also be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

The pharmaceutical composition or unit dosage form of the invention maybe administered according to a dosage and administration regimen definedby routine testing in the light of the guidelines given above in orderto obtain optimal activity while minimizing toxicity or side effects fora particular patient. However, such fine tuning of the therapeuticregimen is routine in the light of the guidelines given herein.

The dosage of the active agents of the invention may vary according to avariety of factors such as underlying disease conditions, theindividual's condition, weight, gender and age, and the mode ofadministration. An effective amount for treating a disorder can easilybe determined by empirical methods known to those of ordinary skill inthe art, for example by establishing a matrix of dosages and frequenciesof administration and comparing a group of experimental units orsubjects at each point in the matrix. The exact amount to beadministered to a patient will vary depending on the state and severityof the disorder and the physical condition of the patient. A measurableamelioration of any symptom or parameter can be determined by a personskilled in the art or reported by the patient to the physician.

The amount of the agent to be administered can range between about 0.01and about 25 mg/kg/day, preferably between about 0.1 and about 10mg/kg/day and most preferably between 0.2 and about 5 mg/kg/day. It willbe understood that the pharmaceutical formulations of the invention neednot necessarily contain the entire amount of the agent that is effectivein treating the disorder, as such effective amounts can be reached byadministration of a plurality of doses of such pharmaceuticalformulations.

In a preferred embodiment of the invention, the compounds according toformula I are formulated in capsules or tablets, preferably containing10 to 200 mg of the compounds of the invention, and are preferablyadministered to a patient at a total daily dose of 10 to 300 mg,preferably 20 to 150 mg and most preferably about 50 mg.

A pharmaceutical composition for parenteral administration contains fromabout 0.01% to about 100% by weight of the active agents of theinvention, based upon 100% weight of total pharmaceutical composition.

Generally, transdermal dosage forms contain from about 0.01% to about100% by weight of the active agents versus 100% total weight of thedosage form.

The pharmaceutical composition or unit dosage form may be administeredin a single daily dose, or the total daily dosage may be administered individed doses. In addition, co-administration or sequentialadministration of another compound for the treatment of the disorder maybe desirable. To this purpose, the combined active principles areformulated into a simple dosage unit.

For combination treatment where the compounds are in separate dosageformulations, the compounds can be administered concurrently, or eachcan be administered at staggered intervals. For example, the compound ofthe invention may be administered in the morning and the antimuscariniccompound may be administered in the evening, or vice versa. Additionalcompounds may be administered at specific intervals too. The order ofadministration will depend upon a variety of factors including age,weight, gender and medical condition of the patient; the severity andaetiology of the disorders to be treated, the route of administration,the renal and hepatic function of the patient, the treatment history ofthe patient, and the responsiveness of the patient. Determination of theorder of administration may be fine-tuned and such fine-tuning isroutine in the light of the guidelines given herein.

DESCRIPTION OF THE INVENTION

Synthesis

Compounds of formula I, and enantiomers, diastereomers, N-oxides, andpharmaceutically acceptable salts thereof, may be prepared by thegeneral methods outlined hereinafter, said methods constituting afurther aspect of the invention.

The compounds of this invention can be prepared by employing reactionsas shown in the following schemes, in addition to other standardmanipulations that are known in the literature, exemplified in theexperimental section or clear to one skilled in the art. The startingmaterials which are not described herein are either commerciallyavailable or may be prepared by employing reactions described in theliterature or are clear to one skilled in the art. The followingexamples are provided so that the invention might be more fullyunderstood, are illustrative only, and should not be construed aslimiting.

It will be appreciated by those skilled in the art that it may bedesirable to use protected derivatives of intermediates used in thepreparation of the compounds according to formula I. Protection anddeprotection of functional groups may be performed by methods known inthe art (see, for example, Green and Wuts Protective Groups in OrganicSynthesis. John Wiley and Sons, New York, 1999).

The abbreviation PG describes a “protecting group” which is introducedto a reactive group before a certain manipulation is carried out, andwhich is later removed. Examples of PG's for protecting a reactive groupinclude: acetyl-, trifluoracetyl-, benzoyl-, ethoxycarbonyl-,N-tert-butoxycarbonyl-(BOC), N-benzyloxycarbonyl-(Cbz), benzyl-,methoxybenzyl-, 2,4-dimethoxybenzyl- and for amino groups additionallythe phthalyl-group for amino-alkylamino or imino groups;N-methoxynethyl-(MOM), N-benzyloxymethyl-(BOM),N-(trimethylsilyl)ethoxymethyl-(SEM),N-tert-butyl-dimethylsiloxymethyl-, N-tert-butyl-dimethylsilyl-(TBDMS),N-triisopropylsilyl-(TIPS), N-benzyl-, N-4-methoxybenzyl (PMB),N-triphenylmethyl-(Tr), N-tert-butoxycarbonyl-(BOC),N-benzyloxycarbonyl-(Cbz) or N-trimethylsilylethylsulfonyl-(SES) foramide groups; methoxy-, benzyloxy-, trimethylsilyl-(TMS), acetyl-,benzoyl-, tert-butyl-, trityl-, benzyl-, or tetrahydropyranyl (THP)groups for hydroxy groups; or trimethylsilyl-(TMS), methyl-ethyl-,tert-butyl-, benzyl-, or tetrahydropyranyl (THP) groups for carboxylgroups.

In some cases the final product may be further modified, for example bymanipulation of substituents. These manipulations may include, but arenot limited to, reduction, oxidation, alkylation, acylation, andhydrolysis reactions, which are commonly known to those skilled in theart. In some cases the order of carrying out the foregoing reactionschemes may be varied in order to facilitate the reaction or to avoidunwanted reaction products. The following examples are provided so thatthe invention might be more fully understood. These examples areillustrative only and should not be constructed as limiting theinvention in any way.

The compounds of the invention are generally prepared according to thefollowing scheme, wherein groups R₁, R₂, R₃, and n are as previouslydefined herein:

As shown in Scheme 1, silyl protected propargylaldehyde oxime 1 isreacted with N-protected unsaturated cyclic amines through a 1,3-dipolarcycloaddition, with the previous formation of the nitrile oxide speciesvia halogenation-elimination (see e.g. Kanemasa, S.; Nishiuchi, M.;Kamimure, A.; Hori, K. J. Am. Chem. Soc. (1994), Vol. 116, pp. 2324).Compounds 2 formed therefrom can be then reacted with an R₃LG compounddirectly, or by deprotection of the alkyne moiety using standardmethodologies (e.g., NaOH or Na₂CO₃ in MeOH, or tetrabutylammoniumfluoride in THF). LG represents a leaving group such as halogen,mesylate, tosylate, alkylsulphonate, triflate or other withoutlimitation. This reaction is performed e.g. by carrying out aSonogashira (Chinchilla et al., Chem. Rev., (2007), Vol. 107 (3), pp.874-922) or like reaction, with the aid of a palladium catalyst andcopper iodide. Following N-deprotection by standard methods, reactionwith an R₂—R₁-LG group follows, where LG is as defined above. This lastderivatization procedure can be done using standard methods such us e.g.Buchwald reactions, acylation reactions, reaction withalkyl/arylisocyanates, alkyl/arylchloroformate, chloroformamides,reductive amination, alkylation or any kind of N-derivatization reactionuseful to the aim of forming compounds according to formula I and verywell known to people skilled in the art. This last reaction can becarried out also by the previous formation of suitable intermediatese.g. a chlorosulphonyl or chlorocarbonyl N-derivative of intermediate 5.

Alternatively, compounds of the invention can be prepared according toScheme 2:

Following Scheme 2, the R₃ group is introduced at the beginning of thesynthetic pathway by a Sonogashira or Sonogashira-like reaction of thedialkyl or cyclic acetal of propiolaldehyde with the proper alkylating,arylating or derivatising reagent R₃-LG where LG is a leaving group asdefined above.

The syntheses of other compounds not currently described in the generaldescription above are well documented inside the experimental part ofthis invention, which follows.

The free bases of compounds according to formula I, their diastereomersor enantiomers can be converted to the corresponding pharmaceuticallyacceptable salts under standard conditions well known in the art. Forexample, the free base is dissolved in a suitable organic solvent, suchas methanol, treated with, for example one equivalent of maleic oroxalic acid, one or two equivalents of hydrochloric acid ormethanesulphonic acid, and then concentrated under vacuum to provide thecorresponding pharmaceutically acceptable salt. The residue can then bepurified by recrystallization from a suitable organic solvent or organicsolvent mixture, such as methanol/diethyl ether.

The N-oxides of compounds according to formula I can be synthesized bysimple oxidation procedures well known to those skilled in the art.

Biological Assay

Cell lines stably transfected were generated using inducible expressionvectors encoding human mGlu₅ receptor using the Tetracycline-RegulatedExpression system (T-REx™ system, Invitrogen, Life Technologies). HumanmGluR₅ open reading frame (ORF), comprehensive of the stop codon, werecloned into the pcDNA4/TO/myc-His™ A vector, carrying the TetO2sequences. The insertion site was HindIII-PstI for mGluR₅ receptors. Theobtained constructs were then transfected into the T-REx CHO™ cell lineusing the FuGENE protocol (Roche); the CHO T-REx™ cell line stablyexpresses the Tet repressor (from the pcDNA6/TR plasmid) under theselection of blasticidin, 10 μg/ml. Stable clones were obtainedselecting with zeocine 1 mg/ml and maintaining in ULTRA CHO medium(LONZA) supplemented with dialyzed FBS, zeocin, blasticidine, at 37° C.,in an atmosphere of 5% CO₂. The expression of h-mGluR₅ receptors wasde-repressed with 1 μg/ml tetracycline for 18 h before bindingexperimentation, while the expression of h-mGluR₅ receptors wasde-repressed respectively with 3 ng/ml and 10 ng/ml tetracycline for 18h before calcium fluorescence experimentation.

Radioligand Binding Assay at Native mGluR₅ and mGluR₅ Receptor Subtypes

Affinity at transmembrane glutamate metabotropic mGluR₅ receptorsubtypes was evaluated according to the methods of Anderson (Anderson etal., J Pharmacol. Exp. Ther., (2002), Vol. 303(3), pp. 1044-51), withsome modifications. Cloned mGluR₅ was obtained by re-suspending CHOT-REx h-mGluR₅ cells (50 μg/well) in 20 mM HEPES, 2 mM MgCl₂, 2 mMCaCl₂, pH 7.4, that then were incubated in a final volume of 1 ml for 60min at 25° C. with 4 nM [³H]MPEP in the absence or presence of competingdrugs. Non-specific binding was determined in the presence of 10 mMMPEP. The incubation was stopped by addition of cold Tris buffer pH 7.4and rapid filtration through 0.2% polyethyleneimine pretreated Filtermat1204-401 (Perkin Elmer) filters. The filters were then washed with coldbuffer and the radioactivity retained on the filters was counted byliquid scintillation spectrometry (Betaplate 1204 BS-Wallac).

Calcium Fluorescence Measurements

Cells were seeded into black-walled, clear-bottom, 96-well plates at adensity of 80000 cell/well, in RPMI (without Phenol Red, withoutL-glutamine; Gibco LifeTechnologies, CA) supplemented with 10% dialyzedFBS. Following 18-h incubation with tetracycline, the cells were loadedwith 2 mM Ca²⁺—sensitive fluorescent dye Fluo-4/AM (Molecular Probes) inHanks' balanced saline solution (HBSS, Gibco LifeTechnologies, CA) with20 mM Hepes (Sigma) and 2.5 mM probenecid (Sigma), for 1 h at 37° C. Thecells were washed three times with HBSS to remove extracellular dye.Fluorescence signals were measured by using the fluorescence microplatereader Flexstation III (Molecular Devices) at sampling intervals of 1.5s for 60 s.

The antagonist potency was determined using the EC₈₀ of the quisqualateused as agonist and the potentiation of mGlu₅ activation was determinedusing the EC₂₀ of the agonist (quisqualate or glutamate). The compoundswere applied 10 minutes before the application of the agonist. Forbinding and calcium assay studies, the compounds were dissolved in DMSOor demineralized water according to their solubility. All the reporteddoses were those of the corresponding salts or bases.

Statistical Analysis.

The inhibition curves of the tested compounds at native and clonedmGluR₁ and mGluR₅ subtypes were determined by nonlinear regressionanalysis using software Prism 4.0 (Graphpad, San Diego, Calif.). TheIC₅₀ values and pseudo-Hill slope coefficients were estimated by theprogram. The values for the inhibition constant, K_(i), were calculatedaccording to the equation K_(i)=IC₅₀/(1+[L]/K_(d)), where [L] is theconcentration of radioligand and K_(d) is the equilibrium dissociationconstant of the radioligand-receptor complex (Cheng et al., Biochem.Pharmacol. (1973), Vol. 22, pp. 3099-3108).

Biological data for selected compounds is presented in Table 1.

TABLE 1 h.c. Fold Fold h.c. mglu mgluR5 Fold Increase 1 EC₅₀ Fold Shift1 R5 IC₅₀ Calcium EC₅₀ nM Increase microM nM Inc. microM Example nM IC₅₀nM Glutam. Glutam. Glutam. Quisq. Quisq. Quisq.  1 3.87 >1000 37.11 1.961.8  2 29.6 >1000 0  3 81.5 54.76  4 6.99  5 707.9 0  6 24.8 95.18 5.13.24 132.15 3.3 2.7  6a 4875.5 0.745  6b 57.01 3.98 3.62  7 59.2 211.8 0 8 12.52 >1000  9 15.02 >1000 10 3.65 >1000 140.7 3.5 2.2 11 17 >1000 1210.6 13 50.03 >1000 130.7 2.06 3.2 14 14.8 >1000 15 21.97 21.1 16 151.617 >1000 1.27 18 >1000 19 239.4 20 >1000 0 21 0 0 22 777 4.7 3.11 23 1442.49 4.73 24 33.94 5.8 8.48 25 0 0 26 54.58 5.09 2.65 27 96.86 5.11 4.0828 0 0 29 0 0 30 108.3 3.69 3.95 31 300.8 5.29 2.14 32 1100 2.92 1.92 3334 541.9 3.31 5.6 36 0 0Preparation of Compounds of the General Formula I

Unless otherwise stated, one or more tautomeric forms of compounds ofthe examples described hereinafter may be prepared in situ and/orisolated. All tautomeric forms of compounds of the examples describedhereinafter should be considered to be disclosed.

The invention is illustrated by way of the following examples, in whichthe following abbreviations may be employed:

AcOH acetic acid

AN acetonitrile

BOC tert-butyloxycarbonyl

conc. concentrated

DCM dichloromethane

DIPEA N,N-diisopropylethylamine

DMF N,N-dimethylformamide

DMSO dimethyl sulfoxide

DPPF 1,1′-bis(diphenyl-phosphino)ferrocene

EI electron ionisation

ESI electrospray ionisation

EtOAc ethyl acetate

EtOH ethanol

HATU 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate

HCl hydrochloric acid

HCOOH formic acid

HPLC high performance liquid chromatography

HPLC-MS HPLC coupled with mass spectrometry

i.vac. under vacuum

MeOH methanol

MS mass spectrometry

MW molecular weight

NaOH sodium hydroxide

NH₄OH ammonium hydroxide (30% ammonia in water)

PE petroleum ether

R_(f) retention value (from thin layer chromatography)

RT room temperature

R.sub.t retention time (from HPLC)

TBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate

THF tetrahydrofurane

TEA triethyl amine

TFA trifluoracetic acid

THF tetrahydrofurane.

The following table (Table 2) illustrates some example compounds of theinvention according to general formula I, that were prepared accordingto Scheme 1 or Scheme 2 accordingly:

Table 2

TABLE 2 Ref. HPLC- Syn- MS thesis Ex. Structure Chemical Name MF MW (M +H)+ Scheme 1

tert-butyl 3-[2-(3- chlorophenyl)ethynyl]- 3a,4,5,6a-tetrahydropyrrolo[3,2- d]isoxazole-6-carboxylate C₁₈H₁₉ClN₂O₃ 346.80347.11 1 2

[3-[2-(3-chlorophenyl)ethynyl]- 3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazol-6-yl]-(2- furyl)methanone C₁₈H₁₃ClN₂O₃ 340.76 341.77 1 3

ethyl 3-[2-(3- chlorophenyl)ethynyl]- 3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate C₁₆H₁₅ClN₂O₃ 318.76 319.76 1 4

3-[2-(3-chlorophenyl)ethynyl]- N-ethyl-N-isopropyl-3a,4,5,6a-tetrahydropyrrolo[3,2- d]isoxazole-6-carboxamide C₁₉H₂₂ClN₃O₂ 359.85360.86 1 5

tert-butyl 3-[2-(6-methyl-2- pyridyl)ethynyl]-3a,4,5,6a-tetrahydropyrrolo[3,2- d]isoxazole-6-carboxylate C₁₈H₂₁N₃O₃ 327.38328.38 2 6

[3-[2-(3-chlorophenyl)ethynyl]- 3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazol-6-yl]-morpholino- methanone C₁₈H₁₈ClN₃O₃ 359.81 360.81 1 7

3-[2-(3-chlorophenyl)ethynyl]- N,N-dimethyl-3a,4,5,6a-tetrahydropyrrolo[3,2- d]isoxazole-6-carboxamide C₁₆H₁₆ClN₃O₂ 317.77318.78 1 8

[3-[2-(3-chlorophenyl)ethynyl]- 3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazol-6-yl]-(1- piperidyl)methanone C₁₉H₂₀ClN₃O₂ 357.84 358.84 1 9

[3-[2-(3-chlorophenyl)ethynyl]- 3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazol-6-yl]-pyrrolidin-1-yl- methanone C₁₈H₁₈ClN₃O₂ 343.81 344.81 110

tert-butyl 3-[2-(m- tolyl)ethynyl]-3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate C₁₉H₂₂N₂O₃ 326.39 327.4 2 11

[3-[2-(m-tolyl)ethynyl]- 3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazol-6-yl]-pyrrolidin-1-yl- methanone C₁₉H₂₁N₃O₂ 323.39 324.4 2 12

[3-[2-(m-tolyl)ethynyl]- 3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazol-6-yl]-(1- piperidyl)methanone C₂₀H₂₃N₃O₂ 337.42 338.42 2 13

morpholino-[3-[2-(m- tolyl)ethynyl]-3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazol-6-yl]methanone C₁₉H₂₁N₃O₃ 339.39 340.4 2 14

N,N-diethyl-3-[2-(m- tolyl)ethynyl]-3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazole-6-carboxamide C₁₉H₂₃N₃O₂ 325.41 326.41 2 15

N-methoxy-N-methyl-3-[2-(m- tolyl)ethynyl]-3a,4,5,6a-tetrahydropyrrolo[3,2- d]isoxazole-6-carboxamide C₁₇H₁₉N₃O₃ 313.36314.36 2 16

ethyl 3-[2-(m-tolyl)ethynyl]- 3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate C₁₇H₁₈N₂O₃ 298.34 299.34 2 17

[3-[2-(6-methyl-2- pyridyl)ethynyl]-3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazol-6-yl]-(1- piperidyl)methanone C₁₉H₂₂N₄O₂ 338.41 339.42 2 18

[3-[2-(6-methyl-2- pyridyl)ethynyl]-3a,4,5,6a- tetrahydropyrrolo[3,2-d]isoxazol-6-yl]-morpholino- methanone C₁₈H₂₀N₄O₃ 340.38 341.37 2 19

N-methoxy-N-methyl-3-[2-(6- methyl-2-pyridyl)ethynyl]- 3a,4,5,6a-tetrahydropyrrolo[3,2- d]isoxazole-6-carboxamide C₁₆H₁₈N₄O₃ 314.34315.35 2 20

ethyl 3-[2-(6-methyl-2- pyridyl)ethynyl]-3a,4,5,6a-tetrahydropyrrolo[3,2- d]isoxazole-6-carboxylate C₁₆H₁₇N₃O₃ 299.33300.33 2

¹H-NMR data for selected compounds above is shown below in Table 3.

TABLE 3 Selected ¹H-NMR data Ex. ¹H-NMR 1 (400 MHz, DMSO-d6) δ ppm 1.44(s, 9 H) 2.20 (br. s., 2H) 2.90-3.09 (m, 1H) 3.61-3.71 (m, 1H) 4.15 (br.s., 1H) 6.18-6.37 (m, 1H) 7.45-7.53 (m, 1H) 7.57-7.63 (m, 2H) 7.74 (s,1H) 2 (400 MHz, CHLOROFORM-d) δ ppm 2.22 (d, 1H) 2.42 (br. s., 1H) 3.33(br. s., 1H) 4.11 (br. s., 1H) 4.38 (dd, 1H) 6.56 (dd, 1H) 6.88 (d, 1H)7.30 (br. s., 1H) 7.32-7.38 (m, 1H) 7.41-7.49 (m, 2H) 7.56 (t, 1H) 7.61(s, 1H) 3 (400 MHz, DMSO-d6) δ ppm 1.22 (t, 3H) 2.23 (br. s., 2H)2.95-3.15 (m, 1H) 3.66-3.76 (m, 1H) 4.05-4.24 (m, 3H) 6.32 (br. s., 1H)7.46-7.53 (m, 1H) 7.56-7.63 (m, 2H) 7.75 (t, 1H) 4 (400 MHz,CHLOROFORM-d) δ ppm 1.16 (t, 3H) 1.22 (t, 6H) 2.18 (tt, 1H) 2.29 (dd,1H) 3.09-3.35 (m, 3H) 3.76 (dd, 1H) 3.92 (t, 1H) 4.13 (spt, 1H) 6.59 (d,1H) 7.30-7.36 (m, 1H) 7.39-7.46 (m, 2H) 7.54 (t, 1H) 6 (400 MHz,CHLOROFORM-d) δ ppm 2.10-2.23 (m, 1H) 2.30 (dd, 1H) 3.18 (td, 1H)3.43-3.50 (m, 4H) 3.73 (m, 4H) 3.87-3.98 (m, 2H) 6.57 (d, 1H) 7.33 (dd,1H) 7.43 (m, 2H) 7.54 (s, 1H) 7 (400 MHz, CHLOROFORM-d) δ ppm 2.08-2.22(m, 1H) 2.25-2.34 (m, 1H) 2.96 (s, 6H) 3.25 (td, 1H) 3.80 (dd, 1H) 3.92(dd, 1H) 6.60 (s, 1H) 7.30-7.37 (m, 1H) 7.38-7.47 (m, 2H) 7.54 (t, 1H) 8(400 MHz, CHLOROFORM-d) δ ppm 1.49-1.71 (m, 6H) 2.09-2.22 (m, 1H)2.23-2.32 (m, 1H) 3.21 (td, 1H) 3.37 (m, 4H) 3.82 (dd, 1H) 3.91 (dd, 1H)6.61 (d, 1H) 7.33 (dd, 1H) 7.38-7.47 (m, 2H) 7.54 (t, 1H) 9 (400 MHz,CHLOROFORM-d) δ ppm 1.80-1.98 (m, 4H) 2.11-2.24 (m, 1H) 2.31 (dd, 1H)3.26 (td, 1H) 3.41-3.49 (m, 2H) 3.49-3.57 (m, 2H) 3.88 (dd, 1H) 3.94(dd, 1H) 6.62 (d, 1H) 7.33 (dd, 1H) 7.38-7.47 (m, 2H) 7.54 (t, 1H) 10(400 MHz, DMSO-d6) δ ppm 1.44 (s, 9 H) 2.09-2.29 (m, 2H) 2.33 (s, 3H)2.86-3.09 (m, 1H) 3.57-3.72 (m, 1H) 4.14 (br. s., 1H) 6.15-6.37 (m, 1H)7.30-7.43 (m, 3H) 7.45 (s, 1H) 11 (400 MHz, CHLOROFORM-d) δ ppm1.79-1.97 (m, 4H) 2.16 (m, 1H) 2.32 (dd, 1H) 2.37 (s, 3H) 3.26 (td, 1H)3.40-3.48 (m, 2H) 3.48-3.59 (m, 2H) 3.87 (dd, 1H) 3.93 (dd, 1H) 6.59 (d,1H) 7.20-7.31 (m, 2H) 7.31-7.40 (m, 2H) 13 (400 MHz, CHLOROFORM-d) δ ppm2.16 (m, 1H) 2.33 (dd, 1H) 2.38 (s, 3H) 3.19 (td, 1H) 3.43-3.50 (m, 4H)3.68-3.77 (m, 4H) 3.86-3.99 (m, 2H) 6.54 (d, 1H) 7.21-7.32 (m, 2H)7.34-7.41 (m, 2H) 14 (400 MHz, CHLOROFORM-d) δ ppm 1.19 (t, 6H)2.10-2.23 (m, 1H) 2.32 (dd, 1H) 2.38 (s, 3H) 3.22 (td, 1H) 3.26-3.43 (m,4H) 3.79 (dd, 1H) 3.91 (dd, 1H) 6.57 (d, 1H) 7.21-7.31 (m, 2H) 7.33-7.40(m, 2H) 16 (400 MHz, DMSO-d6 343 K) δ ppm 1.24 (t, 3H) 2.16-2.25 (m, 2H)2.35 (s, 3H) 2.99-3.10 (m, 1H) 3.65-3.76 (m, 1H) 4.14 (q, 2H) 4.06-4.31(m, 1H) 6.30 (d, 1H) 7.28-7.42 (m, 3H) 7.42-7.46 (m, 1H) 17 (400 MHz,CHLOROFORM-d) δ ppm 1.58-1.70 (m, 6H) 2.08-2.21 (m, 1H) 2.34 (dd, 1H)2.61 (s, 3H) 3.19 (td, 1H) 3.32-3.43 (m, 4H) 3.81 (dd, 1H) 3.94 (dd, 1H)6.60 (d, 1H) 7.20 (d, 1H) 7.41 (d, 1H) 7.62 (t, 1H) 18 (400 MHz,CHLOROFORM-d) δ ppm 2.02-2.22 (m, 1H) 2.35 (dd, 1H) 2.58 (s, 3H) 3.15(td, 1H) 3.35-3.52 (m, 4H) 3.63-3.78 (m, 4H) 3.86 (dd, 1H) 3.96 (dd, 1H)6.55 (d, 1H) 7.19 (d, 1H) 7.39 (d, 1H) 7.61 (t, 1H) 19 (400 MHz,CHLOROFORM-d) δ ppm 2.18 (m, 1H) 2.42 (dd, 1H) 2.63 (s, 3H) 3.15 (s, 3H)3.34 (td, 1H) 3.69 (s, 3H) 3.90 (dd, 1H) 4.01 (dd, 1H) 6.66 (d, 1H) 7.23(d, 1H) 7.43 (d, 1H) 7.65 (t, 1H) 20 (400 MHz, CHLOROFORM-d) δ ppm1.28-1.38 (m, 3H) 2.11-2.28 (m, 1H) 2.40 (dd, 1H) 2.60 (s, 3H) 3.22 (td,1H) 3.73-3.93 (m, 1H) 3.93-4.08 (m, 1H) 4.24 (d, 2H) 6.28-6.52 (m, 1H)7.20 (d, 1H) 7.40 (d, 1H) 7.57-7.66 (m, 1H)

The following examples illustrate some of the compounds of generalformula I as described above. These examples are illustrative only andare not intended to limit the scope of the invention. The reagents andstarting materials are readily available to those skilled in the art.

Example 63-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(morpholin-4-yl)methanone1-chloro-3-(3,3-diethoxyprop-1-ynyl)benzene (Intermediate 6a)

A mixture of 1-chloro-3-iodobenzene (4 g, 16.8 mmol), propargylaldehydediethyl acetal (2.66 mL, 18.5 mmol),bis(trifenilphosphine)palladium(II)dichloride (295 mg, 0.42 mmol),cuprous iodide (160 mg, 0.84 mmol) and triethylamine (60 mL) was stirredat r.t. for 3 h. After 4 h, the reaction mixture was quenched with H₂O,extracted with EtOAc, which was washed with brine, dried over Na₂SO₄,and evaporated to dryness in vacuo. The residue was purified byautomated flash chromatography (Horizon®™—Biotage; PetroleumEther:EtOAc, 97:3) to give 4 g of the title compound as a fluidyellowish oil. Yield: 100%.

MS: [M+H]⁺=239.32.

3-(3-chlorophenyl)prop-2-ynal (Intermediate 6b)

To a solution of Intermediate 6a (4 g, 16.7 mmol) in CH₂Cl₂ (50 mL) wasadded 38.8 mL of water and 7.7 mL of trifluoroacetic acid. After 4 h ofstirring, a further 4 eq. of trifluoroacetic acid was added. After 24 hthe conversion was completed; the 2 layers were separated, the organiclayer was washed with water, dried over Na₂SO₄ and evaporated to drynessin vacuo to afford the title compound as a yellow-brownish oil, used inthe next step without further purification.

MS: [M+H]+=165.35.

3-(3-chlorophenyl)prop-2-ynal oxime (Intermediate 6c)

A mixture of 3-chlorophenylpropargylaldehyde (22.8 g, 139 mmol),hydroxylamine hydrochloride (416 mmol, 28.9 g), EtOH (200 mL) and water(50 mL) was stirred at r.t. for 24 h. The reaction mixture was dilutedwith H₂O, extracted with Et₂O:EtOAc, washed with brine and evaporated todryness in vacuo affording 24 g of the title compound (syn:anti 1:1) asa pasty brownish solid. The pale brown residue was used in the next stepwithout further purification.

Yield: 96.4%.

MS: [M+H]+=180.16.

t-butyl-3-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate(Intermediate 6d)

To a solution of Intermediate 6c (20.68 mmol, 3.72 g) inN,N-dimethylformamide (40 mL) was added N-chlorosuccinimide (23.64 mmol,3.16 g) and the mixture was stirred at room temperature for 2 h. Thenwater was added and the aqueous layer was extracted with Et₂O. Theorganic phase was dried over Na₂SO₄, filtered and evaporated. The cruderesidue was dissolved in CH₂Cl₂ (40 mL) and cooled at 0° C., thentert-butyl 2,3-dihydropyrrole-1-carboxylate (5.91 mmol, 1 g) followed byTEA (17.73 mmol, 1.79 g, 2.47 mL) were added and the mixture was stirredat room temperature overnight. Afterwards, water was added, the twophases were separated, the organic layer was washed with water andbrine, dried over Na₂SO₄. The solvent was removed in vacuo and the cruderesidue was purified via automated flash chromatography (Isolera®Biotage, SNAP100 cartidge) eluting with EtOAc:Petroleum Ether gradientfrom 5% to 50% of EtOAc. The title product (1.1 g) was isolated as abrownish solid.

3-(3-chlorophenylethynyl)-4,5,6,6a-tetrahydropyrrolo[3,2-d]isoxazole(Intermediate 6e)

Into a solution of tert-butyl3-[2-(3-chlorophenyl)ethynyl]-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate(intermediate 6d, 2.88 mmol, 1 g) in CHCl3 (40 mL) stirred at 0° C. wasadded dropwise trifluoroacetic acid (28.84 mmol, 3.288 g, 2.208 mL) andthe mixture was heated at 60° C. for 5 hours. The reaction was checkedby LC/MS showing the correct (M+H)+ peak. The mixture was cooled at 0-5°C. and alkalinized with NaOH to pH=9. Afterwards, water was added, thetwo phases were separated, the organic layer was washed with water andbrine, dried over Na₂SO₄. The solvent was removed in vacuo affording thetitle product (0.7 g, 98.4%) as a brown oil that was used for the nextstep without purification.

3-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(morpholin-4-yl)methanone

To a solution of3-(3-chlorophenylethynyl)-4,5,6,6a-tetrahydropyrrolo[3,2-d]isoxazole(intermediate 6e, 0.6 g, 2.4 mmol) in dichloromethane (40 mL) andtriethylamine (0.63 mL, 2.8 mmol) was added dropwise4-morpholinecarbonyl chloride (0.42 mL, 3.6 mmol) and the resultingmixture was stirred overnight at r.t. Afterwards it was heated at 50° C.for 4 h. The reaction mixture was then poured into water, the organiclayer was separated, dried over Na₂SO₄ and evaporated to dryness invacuo. The crude product was purified by flash chromatography (SP1®Biotage) eluting with a gradient petroleum ether:ethyl acetate 9:1 to6:4 affording the title compound as a white solid (0.45 g, 51% yield).

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.10-2.23 (m, 1H) 2.30 (dd, 1H)3.18 (td, 1H) 3.43-3.50 (m, 4H) 3.73 (m, 4H) 3.87-3.98 (m, 2H) 6.57 (d,1H) 7.33 (dd, 1H) 7.43 (m, 2H) 7.54 (s, 1H).

MS: [M+H]⁺=239.32.

Example 20ethyl-3-[(6-methyl-2-pyridyl)ethynyl]-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylateN-hydroxy-3-trimethylsilyl-prop-2-ynimidoyl chloride (Intermediate 20a)

To a solution of 3-trimethylsilylprop-2-ynal oxime (Carreira, Erick M.;Lohse-Fraefel, Nina, Organic Letters, (2005), Vol. 7, No. 10, pp.2011-2014, 68 g, 11.9 mmol) in 11.9 mL of DMF stirred at r.t. was addedN-chlorosuccinimide (1.99 g, 14.8 mmol). After 4 h stirring, thesolution was poured into water and extracted with Et₂O. After the usualwork-up, the residue (2.09 g) was used as it was for the next step.

t-butyl-3-[(trimethylsilyl)ethynyl]-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate(Intermediate 20b)

A solution of TEA (0.554 mL, 3.85 mmol) in 9.4 mL of dichloromethane wasadded dropwise into a solution of Compound 20a (1.67 g, 2.57 mmol) andtert-butyl 2,3-dihydropyrrole-1-carboxylate (600 mg, 2.57 mmol) in 42 mLof dichloromethane stirred at 0° C. Afterwards, the reaction mixture wasstirred at r.t. for 24 h; then it was diluted with cold water. Theorganic layer was washed with brine, dried over Na₂SO₄, evaporated todryness in vacuo. The crude product was purified by automated flashchromatography (SP1®™—Biotage; gradient Petroleum Ether:EtOAc from 5:5to 0:10) affording 641 mg of the title product. Yield: 67%.

t-butyl-3-[(6-methylpyridin-2-yl)ethynyl]-3a,4,5,6a-tetrahydro-pyrrolo[3,2-d]isoxazole-6-carboxylate(Intermediate 20c)

To a solution of Intermediate 20b (200 mg, 0.65 mmol) and2-bromo-6-methylpyridyne (81.1 μl, 0.72 mmol) in N,N-dimethylformamide(4 mL) degassed with a nitrogen stream for 5 min., were added quickly inthe order tetrakis(triphenylphosphine)palladium(0) (22.5 mg, 0.02 mmol),tetrabutylammonium fluoride (186 mg, 0.713 mmol) and sodium acetate (106mg, 1.3 mmol). The mixture was heated in a microwave oven at 120° C. for10 min. The reaction was poured into water and extracted with ethylacetate. The organic layer was dried over Na₂SO₄ and evaporated todryness. The crude product was purified by automated flashchromatography (SPI®™—Biotage) with a gradient petroleum ether:ethylacetate from 8:2 to 3:7. The title product was isolated as a brownishoil (212 mg, 54.2%).

3-[(6-methylpyridin-2-yl)ethynyl]-4,5,6,6a-tetrahydropyrrolo[3,2-d]isoxazole(Intermediate 20d)

The title compound was synthesized using the method reported above forIntermediate 6e, but replacing Intermediate 20c for Intermediate 6d.After the usual work-up procedure the residue was purified by means ofautomated flash chromatography (Horizon®™—Biotage; gradient PetroleumEther:EtOAc from 98:2 to 9:1) to give the title compound. Yield: 95.9%.

ethyl-3-[(6-methyl-2-pyridyl)ethynyl]-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate

To a solution of Intermediate 20d (60 mg, 0.26 mmol) in CH₂Cl₂ (6 mL)was added TEA (0.08 mL) and then, dropwise, ethyl chloroformate (38.1μL, 0.4 mmol). The reaction mixture was stirred at r.t. for 1 h.Afterwards, it was poured into water and extracted with ethyl acetate.The organic layer was dried over Na₂SO₄ and evaporated to dryness. Thecrude product was purified by automated flash chromatography(SP1®™—Biotage) with a gradient petroleum ether:ethyl acetate from 9:1to 4:6. The title product was isolated as a brownish oil which wasfurther purified by preparative HPLC affording the title product. Yield:25.3%.

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.28-1.38 (m, 3H) 2.11-2.28 (m, 1H)2.40 (dd, 1H) 2.60 (s, 3H) 3.22 (td, 1H) 3.73-3.93 (m, 1H) 3.93-4.08 (m,1H) 4.24 (d, 2H) 6.28-6.52 (m, 1H) 7.20 (d, 1H) 7.40 (d, 1H) 7.57-7.66(m, 1H).

Alternative Procedure for the Synthesis of Intermediate 20ct-butyl-3-ethynyl-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate(Intermediate 20e)

To a solution of Intermediate 20b (530 mg, 1.72 mmol) in MeOH (20 mL)was added K₂CO₃ (713 mg, 5.16 mmol) and the mixture was stirred for 1 hat r.t., checked by HPLC-MS, poured into water and extracted with EtOAc.The title compound was obtained by purification with automated flashcolumn chromatography (SP1®™—Biotage) with a gradient petroleumether:ethyl acetate from 7:3 to 6:4. Colourless oil (406 mg, 49.2%).

t-butyl-3-[(6-methylpyridin-2-yl)ethynyl]-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate(Intermediate 20c)

To a solution of Intermediate 20e (200 mg, 0.85 mmol) and2-bromo-6-methylpyridyne (106 μl, 0.93 mmol) in N,N-dimethylformamide (4mL) degassed with a nitrogen stream for 5 min., were added quickly inthe order tetrakis(triphenylphosphine)palladium(0) (29.3 mg, 0.025 mmol)and sodium acetate (139 mg, 1.7 mmol) and the mixture was heated in amicrowave oven at 120° C. for 10 min. The reaction was poured into waterand extracted with ethyl acetate. The organic layer was dried overNa₂SO₄ and evaporated to dryness. The crude product was purified byautomated flash chromatography (SP1®™—Biotage) with a gradient petroleumether:ethyl acetate from 8:2 to 3:7. The title product was isolated as abrownish oil (212 mg, 54.2%).

Starting from Intermediate 6e (as hydrochloride) the following compoundswere prepared as follows:

Example 21isopropyl-3-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate

Intermediate 6e (30 mg, 0.1 mmol) was dissolved in DCM (0.6 mL) under anargon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (21 μL, 0.15 mmol) followed by isopropyl chloroformate 1.0M in toluene (127 μL, 0.12 mmol) were added. Stirring was continued atroom temperature overnight. Water was added (5 mL) and the reactionmixture was extracted with DCM (10 mL, 3×). The organic layer was driedover MgSO₄ and evaporated to dryness under reduced pressure to give 55mg of crude product. The crude product was purified by preparative TLC(Hex:EtOAc 6:4), taking up the silica with 5% MeOH in EtOAc. Thefiltrate was concentrated under vacuum to give 11.4 mg (38% yield) ofthe title product.

MS: [M+H]⁺=333.1, [2M+Na]=687.3;

¹H NMR (400 MHz, DMSO-d₆) δ 7.77-7.73 (m, 1H), 7.63-7.57 (m, 2H),7.53-7.47 (m, 1H), 6.35-6.26 (m, 1H), 4.89-4.78 (m, 1H), 4.21-4.12 (m,1H), 3.73-3.65 (m, 1H), 3.08-2.97 (m, 1H), 2.26-2.18 (m, 2H), 1.23 (d,6H).

Example 22cyclopropylmethyl-3-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate

Intermediate 6e (30 mg, 0.1 mmol) was dissolved in DCM (0.6 mL) under anargon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (21 μL, 0.15 mmol) followed by cyclopropylmethylchloroformate (17 mg, 0.12 mmol)) were added. Stirring was continued atroom temperature overnight. Water was added (5 mL) and the reactionmixture was extracted with DCM (10 mL, 3×). The organic layer was driedover MgSO₄, and evaporated to dryness under reduced pressure to give 48mg of crude product. The crude product was purified by preparative TLC(Hex:EtOAc 4:6), taking up the silica with 5% MeOH in EtOAc. Thefiltrate was concentrated under vacuum to give 18.6 mg (50% yield) ofthe title product.

MS: [M+H]⁺=345.8

¹H NMR (400 MHz, DMSO-d₆) δ 7.77-7.74 (m, 1H), 7.64-7.56 (m, 2H), 7.51(dd, 1H), 6.33 (d, 1H), 4.23-4.11 (m, 2H), 3.76-3.67 (m, 1H), 3.14-2.97(m, 2H), 2.29-2.16 (m, 2H), 1.21-1.07 (m, 1H), 0.53 (d, 2H), 0.35-0.26(m, 2H).

Example 23cyclopentyl-3-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate

Intermediate 6e (30 mg, 0.11 mmol) was dissolved in DCM (0.6 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (21 μL, 0.15 mmol)) followed by cyclopentyl chloroformate(16 μL, 0.13 mmol) were added. Stirring was continued for 1 hour at roomtemperature. Water was added (5 mL) and the reaction mixture wasextracted with EtOAc (10 mL, 3×). The organic layer was dried overNa₂SO₄, and evaporated to dryness under reduced pressure to give 54 mgof crude product. The crude product was purified by flash columnchromatography on silica using EtOAc:DCM:Hex 3:1:1 as an eluent. 27 mgof the title compound as a yellow thick oil was obtained (71% yield).

MS: [2M+Na]=739.4

¹H NMR (400 MHz, DMSO-d6) δ 7.78-7.73 (m, 1H), 7.63-7.57 (m, 2H),7.53-7.47 (m, 1H), 6.29 (dd, 1H), 5.05 (s, 1H), 4.26-4.11 (m, 1H),3.73-3.62 (m, 1H), 3.14-2.95 (m, 1H), 2.27-2.15 (m, 2H), 1.90-1.75 (m,2H), 1.75-1.50 (m, 6H).

Example 242,2-dimethylpropyl-3-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropvrrolo[3,2-d]isoxazole-6-carboxylate

Intermediate 6e (30 mg, 0.11 mmol) was dissolved in DCM (0.6 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (21 μL, 0.15 mmol) followed by neopentyl chloroformate (19μL, 0.13 mmol) were added. Stirring was continued for 1 hour at roomtemperature. Water was added (5 mL) and the reaction mixture wasextracted with EtOAc (10 mL, 4×). The organic layer was dried overNa₂SO₄, and evaporated to dryness under reduced pressure to give 48 mgof crude product. The crude product was purified by flash columnchromatography on silica using EtOAc:DCM:Hex 3:1:1 as an eluent. 33 mgof the title compound as a yellow thick oil was obtained (86% yield).

MS: [2M+Na]=743.6

¹H NMR (400 MHz, DMSO-d6) δ 7.76 (s, 1H), 7.66-7.56 (m, 2H), 7.54-7.45(m, 1H), 6.40-6.26 (m, 1H), 4.28-4.10 (m, 1H), 3.89-3.62 (m, 3H),3.22-2.95 (m, 1H), 2.24 (s, 2H), 0.97-0.90 (m, 9H).

Example 253-(3-chlorophenylethynyl)-N-(propan-2-yl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxamide

Intermediate 6e (30 mg, 1.1 mmol) was dissolved in DCM (0.45 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (32 μL, 0.23 mmol)) followed by isopropyl isocyanate (10μL, 0.11 mmol) were added. Stirring was continued for 24 hours at roomtemperature. Water was added (5 mL) and the reaction mixture wasextracted with DCM (10 mL, 3×). The organic layer was dried over MgSO₄and evaporated to dryness under reduced pressure to give 41 mg of crudeproduct. The residue was purified via preparative HPLC to afford 23 mgof the title compound (66% yield).

MS: [M+H]⁺=332.1

¹H NMR (400 MHz, DMSO-d6) δ 7.75 (t, 1H), 7.63-7.56 (m, 2H), 7.53-7.46(m, 1H), 6.44 (d, 1H), 6.35 (d, 1H), 4.14-4.06 (m, 1H), 3.84-3.75 (m,1H), 3.70-3.61 (m, 1H), 3.00-2.89 (m, 1H), 2.24-2.10 (m, 2H), 1.09 (dd,6H).

Example 26N-t-butyl-3-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxamide

Intermediate 6e (30 mg, 0.1 mmol) was dissolved in DCM (0.45 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (49 μL, 0.35 mmol) followed by tert-butyl isocyanate (17μL, 0.14 mmol) were added. Stirring was continued for 24 hours at roomtemperature. Water was added (5 mL) and the reaction mixture wasextracted with DCM (10 mL, 3×). The organic layer was dried over MgSO₄and evaporated to dryness under reduced pressure to give 45 mg of crudeproduct. The residue was purified via flash column chromatography onsilica eluting with AcOEt:Hex 1:1. The collected combined fractions weretaken up with hexane and finally purified via preparative TLC (AcOEt:Hex1:9), taking up the silica with 5% MeOH in EtOAc. The filtrate wasconcentrated under vacuum to give 14 mg (33% yield) of the titleproduct.

MS: [M+H]⁺=346

¹H NMR (400 MHz, DMSO-d6) δ 7.75 (t, 1H), 7.62-7.57 (m, 2H), 7.53-7.47(m, 1H), 6.48 (d, 1H), 5.82 (s, 1H), 4.14-4.05 (m, 1H), 3.69-3.60 (m,1H), 3.03-2.90 (m, 1H), 2.22-2.11 (m, 2H), 1.29 (s, 9H).

Example 273-(3-chlorophenylethynyl)-N-cyclopentyl-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxamide

Intermediate 6e (30 mg, 0.1 mmol) was dissolved in DCM (0.45 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (35 μL, 0.25 mmol) followed by cyclopentyl isocyanate (14μL, 0.12 mmol) were added. Stirring was continued for 24 hours at roomtemperature. Water was added (5 mL) and the reaction mixture wasextracted with DCM (10 mL, 3×). The organic layer was dried over MgSO₄and evaporated to dryness under reduced pressure to give 48 mg of crudeproduct. The crude product was purified via flash column chromatographyon silica eluting with a gradient from Hex:EtOAc 9:1 to EtOAc. Thecollected combined fractions were evaporated to dryness, taken up withhexane, purified by preparative TLC (Hex:EtOAc 9:1) and finally bypreparative HPLC to afford 14 mg (36% yield) of the title product.

MS:[M+H]⁺=358.1

¹H NMR (400 MHz, DMSO-d6) δ 7.77-7.73 (m, 1H), 7.59 (m, 2H), 7.53-7.46(m, 1H), 6.46 (d, 1H), 6.41 (d, 1H), 4.10 (t, 1H), 4.00-3.88 (m, 1H),3.71-3.62 (m, 1H), 3.00-2.89 (m, 1H), 2.23-2.09 (m, 2H), 1.86-1.74 (m,2H), 1.68-1.60 (m, 2H), 1.53-1.37 (m, 4H).

Example 283-(3-chlorophenyl)ethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(furan-3-yl)methanone

Intermediate 6e (30 mg, 0.1 mmol) was dissolved in DCM (0.6 mL) under anargon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (21 μL, 0.14 mmol) followed by furan-3-carbonyl chloride(17 mg, 0.12 mmol) were added. Stirring was continued at roomtemperature overnight. Water was added (5 mL) and the reaction mixturewas extracted with DCM (10 mL, 3×). The organic layer was dried overMgSO₄, and evaporated to dryness under reduced pressure to give 53 mg ofcrude product. The crude product was purified by flash columnchromatography on silica followed by preparative TLC (Hex:EtOAc 2:8)taking up the silica with 5% MeOH in EtOAc. The filtrate was evaporatedunder vacuum to give 22.4 mg of the title product (62% yield).

MS: [M+H]+=341.5

¹H NMR (400 MHz, DMSO) δ 8.30-8.14 (m, 1H), 7.86-7.74 (m, 2H), 7.66-7.57(m, 2H), 7.51 (t, 1H), 6.85-6.78 (m, 1H), 6.69-6.54 (m, 1H), 4.36-4.07(m, 2H), 3.14-2.98 (m, 1H), 2.38-2.15 (m, 2H).

Example 293-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(5-methylfuran-2-yl)methanone

Intermediate 6e (30 mg, 0.1 mmol) was dissolved in DCM (0.6 mL) under anargon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (21 μL, 0.14 mmol) followed by 5-methylfuran-2-carbonylchloride (18 mg, 0.13 mmol) were added. Stirring was continued for 1hour at room temperature. Water was added (5 mL) and the reactionmixture was extracted with EtOAc (10 mL, 3×). The organic layer wasdried over Na₂SO₄ and evaporated to dryness under reduced pressure togive 50 mg of crude product. The crude product was purified by flashcolumn chromatography on silica using EtOAc:DCM:Hex 3:1:1 as an eluent.30 mg of the title compound as a yellow thick oil was obtained (80%yield).

MS: [M+H]⁺=355.1, [2M+Na]=731.4

¹H NMR (400 MHz, DMSO-d6) δ 7.78 (t, 1H), 7.66-7.56 (m, 2H), 7.55-7.46(m, 1H), 7.11 (d, 1H), 6.81 (s, 1H), 6.33 (dd, 1H), 4.30 (s, 1H),4.21-3.86 (m, 2H), 3.08 (s, 1H), 2.36 (s, 3H), 2.30 (s, 1H).

Example 303-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(cyclopentyl)methanone

Intermediate 6e (30 mg, 0.1 mmol) was dissolved in DCM (0.6 mL) under anargon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (37 μL, 0.26 mmol) followed by cyclopentanecarbonylchloride (15 μL, 0.13 mmol) were added. Stirring was continued for 1hour at room temperature. Water was added (5 mL) and reaction mixturewas extracted with DCM (10 mL, 3×). The organic layer was dried overMgSO₄, and evaporated to dryness under reduced pressure to give 46 mg ofcrude product. The crude product was purified by flash columnchromatography on silica using hexane:EtOAc 7:3 as an eluent to afford35 mg of the title product (96% yield).

MS: [M+H]⁺=343.1

¹H NMR (400 MHz, DMSO-d6, mixture of rotamers) δ 7.78-7.75 (m, 1H),7.64-7.58 (m, 2H), 7.50 (t, 1H), 6.60 (d, 1H_(major rotamer)), 6.45 (d,1H_(minor rotamer)), 4.28 (t, 1H_(major rotamer)), 4.13 (t,1H_(minor rotamer)), 3.92-3.82 (m, 1H), 3.24-3.14 (m,1H_(major rotamer)), 3.09-3.00 (m, 1H_(minor rotamer)), 2.99-2.87 (m,1H), 2.29-2.11 (m, 2H), 1.93-1.77 (m, 2H), 1.73-1.52 (m, 6H).

Example 313-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrro[3,5,6a-tetrahdrorrolo3,2-d]oxazol-6-yl-(oxan-4-yl)methanone

Intermediate 6e (30 mg, 0.1 mmol) was dissolved in DCM (0.6 mL) under anargon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (37 μL, 0.26 mmol) followed bytetrahydro-2H-pyran-4-carbonyl chloride (19 mg, 0.13 mmol) were added.Stirring was continued for 1 hour at room temperature. Water was added(5 mL) and reaction mixture was extracted with DCM (10 mL, 3×). Theorganic layer was dried over MgSO₄, and evaporated to dryness underreduced pressure to give 65 mg of crude product. The crude product waspurified by flash column chromatography on silica using a gradientDCM:EtOAc 8:2 to 6:4 as an eluent. 32 mg of the title compound wereobtained (84% yield).

MS:[M+H]⁺=359.1

¹H NMR (400 MHz, DMSO-d6, mixture of rotamers) δ 7.78-7.75 (m, 1H),7.64-7.57 (m, 1H), 7.51 (t, 1H), 6.67 (d, 1H_(major rotamer)), 6.46 (d,1H_(minor rotamer)), 4.29 (t, 1H_(major rotamer)), 4.13 (t,1H_(minor rotamer)), 3.94-3.77 (m, 4H), 3.44-3.33 (m, 3H), 3.00-2.87 (m,1H), 2.35-2.08 (m, 2H), 1.71-1.52 (m, 4H).

Example 323-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(4-methylpiperazin-1-yl)methanone

Intermediate 6e (30 mg, 0.1 mmol) was dissolved in DCM (0.6 mL) under anargon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (37 μL, 0.26 mmol) followed by4-methyl-1-piperazinecarbonyl chloride (17 μL, 0.13 mmol) were added.Stirring was continued for 1 hour at room temperature. Water was added(5 mL) and reaction mixture was extracted with DCM (10 mL, 3×). Theorganic layer was dried over MgSO₄ and evaporated to dryness underreduced pressure to give 42 mg of crude product. The crude product waspurified by flash column chromatography on silica using DCM:MeOH 19:1 asan eluent. 20 mg of the title product were obtained (51% yield).

MS: [M+H]⁺=373.3

¹H NMR (400 MHz, DMSO-d6) δ 7.77-7.73 (m, 1H), 7.62-7.57 (m, 2H),7.53-7.46 (m, 1H), 6.52 (d, 1H), 4.12 (t, 1H), 3.59 (dd, 1H), 3.36-3.28(m, 2H, the signal is partially covered by water), 3.28-3.19 (m, 2H),3.16-3.07 (m, 1H), 2.36-2.24 (m, 4H), 2.23-2.16 (m, 4H), 2.15-2.05 (m,1H).

Example 334-oxanyl-3-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate

Intermediate 6e (30 mg, 0.11 mmol) was dissolved in DCM (0.6 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (21 μL, 15 mmol) followed by oxan-4-yl chloroformate (21mg, 0.13 mmol) were added. Stirring was continued for 1.5 hour at roomtemperature. Water was added (5 mL) and reaction mixture was extractedwith EtOAc (10 mL, 3×). The organic layer was dried over Na₂SO₄ andevaporated to dryness under reduced pressure to give 60 mg of crudeproduct. The crude product was purified by flash column chromatographyon silica using EtOAc:DCM 3:1 as an eluent. After a further flashpurification 28 mg of the title compound as a yellow thick oil wereobtained (70% yield).

MS: [2M+Na]⁺=771.3

¹H NMR (400 MHz, DMSO-d6) δ 7.75 (s, 1H), 7.65-7.56 (m, 2H), 7.53-7.47(m, 1H), 6.35 (t, 1H), 4.83 (s, 1H), 4.20 (s, 1H), 3.87-3.63 (m, 3H),3.50 (d, 2H), 3.05 (s, 1H), 2.24 (s, 2H), 1.87 (s, 2H), 1.59 (s, 2H).

Example 343-methylbutyl-3-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate

Intermediate 6e (30 mg, 0.11 mmol) was dissolved in DCM (0.6 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (21 μL, 0.15 mmol) followed by 3-methylbutyl chloroformate(19 mg, 0.13 mmol) were added. Stirring was continued for 1 hour at roomtemperature. Water was added (5 mL) and reaction mixture was extractedwith EtOAc (10 mL, 3×). The organic layer was dried over Na₂SO₄, andevaporated to dryness under reduced pressure to give 53 mg of crudeproduct. The crude product was purified by column chromatography onsilica using EtOAc:DCM 3:1 as an eluent. 34 mg of the title product as ayellow thick oil was obtained (89% yield).

MS: [2M+Na]⁺=743.3

¹H NMR (400 MHz, DMSO-d6) δ 7.76 (s, 1H), 7.64-7.56 (m, 2H), 7.55-7.46(m, 1H), 6.37-6.23 (m, 1H), 4.24-4.14 (m, 1H), 4.16-4.05 (m, 2H),3.77-3.61 (m, 1H), 3.12-2.97 (m, 1H), 2.22 (m, 2H), 1.79-1.58 (m, 1H),1.49 (dt, 2H), 0.91 (d, 6H).

Example 353-(3-chlorophenylethynyl)-N-(pentan-3-yl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxamide

Intermediate 6e (40 mg, 0.14 mmol) was dissolved in DCM (0.45 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (54 μL, 0.038 mmol) followed by 2-ethylpropylisocyanate(18 mg, 0.15 mmol) were added. Stirring was continued for 24 hours atroom temperature. Water was added (5 mL) and reaction mixture wasextracted with DCM (10 mL, 3×). The organic layer was dried over MgSO₄and evaporated to dryness under reduced pressure to give 57 mg of crudeproduct. The crude product was purified via preparative HPLC to afford42 mg of the title product (78% yield).

MS: [M+H]⁺=360.0

¹H NMR (400 MHz, DMSO-d6) δ 7.75 (t, 1H), 7.63-7.56 (m, 2H), 7.54-7.46(m, 1H), 6.48 (d, 1H), 6.19 (d, 1H), 4.17-4.07 (m, 1H), 3.73-3.62 (m,1H), 3.50-3.41 (m, 1H), 3.03-2.92 (m, 1H), 2.24-2.13 (m, 2H), 1.52-1.31(m, 4H), 0.83 (td, 6H).

Example 363-(3-chlorophenylethynyl)-N-(pyridin-3-yl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxamide

Intermediate 6e (40 mg, 0.14 mmol) was dissolved in DCM (0.8 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (47 μL, 0.33 mmol) followed by pyridine-3-isocyanate (19mg, 0.15 mmol) were added. Stirring was continued for 24 hours at roomtemperature. Water was added (5 mL) and the reaction mixture wasextracted with DCM (10 ml, 3×). The organic layer was dried over MgSO₄,and evaporated to dryness under reduced pressure to give 61 mg of crudeproduct. The crude product was purified via flash column chromatographyon silica with a gradient hexane to Hex:EtOAc 1:1 as eluent. Thecombined collected fractions were evaporated to dryness and furtherpurified by preparative TLC (Hex:EtOAc 6:4). 40 mg of the title compoundwere obtained (77% yield).

MS: [M+H]⁺=367.2, [2M+Na]⁺=755.2

¹H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.70 (d, 1H), 8.22 (dd, 1H),7.94 (ddd, 1H), 7.77 (t, 1H), 7.65-7.58 (m, 2H), 7.55-7.48 (m, 1H), 7.32(dd, 1H), 6.60 (d, 1H), 4.24 (t, 1H), 3.91-3.82 (m, 1H), 3.21-3.10 (m,1H), 2.37-2.24 (m, 2H).

Example 373-(3-chlorophenylethynyl)-N-(2,2-dimethylpropyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxamide

Intermediate 6e (30 mg, 0.1 mmol) was dissolved in DCM (0.45 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (32 μL, 0.24 mmol) followed by 2,2-dimethylpropylisocyanate (12 mg, 0.1 mmol) were added. Stirring was continued for 24hours at room temperature. Water was added (5 mL) and the reactionmixture was extracted with DCM (10 ml, 3×). The organic layer was driedover MgSO₄ and evaporated to, dryness under reduced pressure to give 39mg of crude product. The crude product was purified via preparative HPLCto yield 23 mg of the title product (60%).

MS: [M+H]⁺=360.2

¹H NMR (400 MHz, DMSO-d6) δ 7.75 (t, 1H), 7.63-7.56 (m, 2H), 7.53-7.47(m, 1H), 6.53-6.45 (m, 2H), 4.16-4.09 (m, 1H), 3.71-3.64 (m, 1H),3.07-2.96 (m, 2H), 2.78 (dd, 1H), 2.23-2.16 (m, 2H), 0.84 (s, 9H).

Example 383-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(1,5-dimethyl-1H-pyrazol-3-yl)methanone

Intermediate 6e (50 mg, 0.18 mmol) was suspended in DCM (1 mL) at roomtemperature. TEA (52 μl, 37 mmol) was added and the suspension became aclear, yellow solution. 1,5-dimethyl-1H-pyrazole-3-carbonyl chloride (28mg, 18 mmol) as solid was added. The reaction solution was stirred atroom temperature for 2 h. The solvent was removed in vacuo. Theby-products were removed by dissolving the crude product in MeOH andprecipitating with Et₂O. Afterwards, the solvents were removed in vacuoand the residue was dissolved in 10 mL of EtOAc and washed three timeswith 1M KHSO₄ to give the desired product (34 mg, 53% yield).

MS:[M+H]⁺=369.1, [2M+H]⁺=759.2

¹H NMR (400 MHz, DMSO-d6) δ 7.77 (t, 1H), 7.66-7.56 (m, 2H), 7.50 (t,1H), 7.34 (d, 1H), 6.50 (d, 1H), 4.30 (t, 1H), 4.01 (dd, 1H), 3.80 (d,3H), 3.06 (td, 1H), 2.36-2.10 (m, 5H).

Example 393-(3-chlorophenlethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(thiazol-4-yl)methanone

Intermediate 6e (50 mg, 18 mmol)) was suspended in DCM (1 mL) at roomtemperature. TEA (52 μl, 0.37 mmol)) was added and the suspension becamea clear, yellow solution. Next, 1,3-thiazole-4-carbonyl chloride (26 mg,0.18 mmol) as a solid was added. The reaction solution was stirred atroom temperature for 2 h. The solvent was removed in vacuo and the crudeproduct was purified using flash column chromatography Hex:EtOAc 1:1,TLC_(Rf)=0.24). Trituration with Et₂O increased the product purity from91% to 93%. The final purification step was performed on the prep. HPLC,which yielded 11 mg of the desired product (as formate salt) with apurity of 99.7% (17% yield).

MS: [M+H]⁺=358.1

¹H NMR (400 MHz, DMSO-d6) δ 9.30-9.16 (m, 1H), 8.44 (d, 1H), 7.78 (t,1H), 7.61 (tt, 2H), 7.55-7.45 (m, 1H), 7.35 (d, 1H), 4.34-4.23 (d, 1H),4.03 (dd, 1H), 3.15 (dd, 1H), 2.32-2.22 (m, 2H).

Example 403-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(4,4-difluorocyclohexyl)methanone

Intermediate 6e (30 mg, 0.11 mmol) was dissolved in DCM (0.6 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (37 μL, 26 mmol) followed by4,4-difluorocyclohexane-1-carbonyl chloride (23 mg, 0.13 mmol) wereadded. Stirring was continued at room temperature overnight. Water wasadded (5 mL) and the reaction mixture was extracted with DCM (10 mL,3×). The organic layer was extracted with brine and evaporated todryness under reduced pressure. The crude product was purified by flashcolumn chromatography on silica using DCM:MeOH 95:5 as an eluent to give38.0 mg. The obtained product was further purified by preparative TLC,using as an eluent AcOEt:hexane (1:1) and finally by preparative HPLC togive 17 mg of the title compound (41% yield).

MS: [2M+Na]+=807.3

¹H NMR (400 MHz, DMSO-d6) δ 7.78-7.75 (m, 1H), 7.64-7.58 (m, 2H),7.54-7.48 (m, 1H), 6.65 (d, 1H_(major rotamer)), 6.45 (d,1H_(minor rotamer)), 4.36-4.27 (m, 1H_(major rotamer)), 4.17-4.10 (m,1H_(minor rotamer)), 3.96-3.89 (m, 1H_(minor rotamer)), 3.89-3.80 (m,1H_(major rotamer)), 2.95 (td, 1H), 2.88-2.79 (m, 1H), 2.31-2.20 (m,2H), 2.18-2.00 (m, 2H), 1.98-1.75 (m, 4H), 1.69-1.52 (m, 2H).

Example 413-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(1methyl-piperidin-4-yl)methanone

Intermediate 6e (30 mg, 0.11 mmol) was dissolved in DCM (0.3 mL) underan argon atmosphere. Catalytic DMF was added, then1-methylpiperidine-4-carboxylic acid (30 mg, 0.21 mmol) and DIPEA (55μL, 32 mmol). Stirring was continued for 15 mins at room temperature.After 15 min HATU (85 mg, 0.22 mmol) was added and stirring wascontinued overnight at room temperature. Sodium bicarbonate saturatedaq. solution was added (5 mL) and the reaction mixture was extractedwith DCM (10 mL, 3×). The organic layer was evaporated to dryness underreduced pressured. The crude product was purified by flash columnchromatography on silica using as an eluent DCM:MeOH 9:1 to give 20 mgof solid title compound (51% yield).

MS: [M+H]⁺=372.3

¹H NMR (400 MHz, DMSO-d6) δ 7.78-7.74 (m, 1H), 7.64-7.58 (m, 2H),7.54-7.47 (m, 1H), 6.64 (d, 1H_(major rotamer)), 6.45 (d,1H_(minor rotamer)), 4.35-4.28 (m, 1H_(major rotamer)), 4.18-4.11 (m,1H_(minor rotamer)), 3.96-3.80 (m, 1H), 3.27-3.00 (m, 3H), 3.00-2.89 (m,1H), 2.84-2.71 (m, 1H), 2.31-2.23 (m, 1H), 2.21-2.08 (m, 1H), 1.92-1.58(m, 4H), 1.21-1.11 (m, 1H). Signal from CH₃ group covered by DMSO-d₆.

Example 423-(3-chlorophenylethynyl)-N-(2-methoxyethyl)-N-methyl-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxamide3-(3-chlorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carbonylchloride (Intermediate 42a)

Triphosgene (18 mg, 0.06 mmol) was dissolved in dry DCM (0.4 mL) underan argon atmosphere. The reaction mixture was cooled to 0° C. andpyridyne (14 L, 0.18 mmol) was added. After 5 min Intermediate 6e (50mg, 0.18 mmol) dissolved in dry DCM was added slowly. The reactionmixture was warmed up to room temperature. Stirring was continued for 2hours at room temperature. The reaction was quenched with 1M HCl (0.35mL), extracted 5× with DCM (10 mL), and washed with a saturated aq.solution of NaHCO₃ (5 mL). The organic layer was dried over Na₂SO₄,concentrated and dried under reduced pressure to give 73 mg of crudeproduct. The crude product was purified by flash column chromatographyusing EtOAc:DCM 3:1 as an eluent. 35 mg of a yellow thick oil wasobtained. The product was used immediately in the next step withoutfurther purification.

3-(3-chlorophenylethynyl)-N-(2-methoxyethyl)-N-methyl-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxamide

Intermediate 42a (35 mg, 11 mmol) was dissolved in DCM (0.6 mL) under anargon atmosphere. The reaction mixture was cooled to 0° C. andtriethylamine (32 μL, 0.23 mmol) followed by (2-methoxyethyl)methylamine(25 μL, 0.23 mmol) were added. Stirring was continued for 2 hours atroom temperature. Water was added (5 mL) and the reaction mixture wasextracted with DCM (10 mL, 3×). The organic layer was dried over Na₂SO₄and evaporated to dryness under reduced pressure to give 53 mg of crudeproduct. The crude product was purified by flash column chromatographyon silica using EtOAc:DCM 3:1 as an eluent. 25 mg of yellow thick oilwas obtained (61% yield).

MS: [2M+Na]⁺=745.3

¹H NMR (400 MHz, DMSO-d6) δ 7.75 (t, 1H), 7.64-7.55 (m, 2H), 7.54-7.46(m, 1H), 6.52 (d, 1H), 4.12 (t, 1H), 3.61-3.39 (m, 4H), 3.31-3.27 (m,1H), 3.26 (s, 3H), 3.18-3.05 (m, 1H), 2.88 (s, 3H), 2.25-2.01 (m, 2H).

Example 433-(3-fluorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-morpholin-4-yl-methanonetert-butyl-(3-trimethylsilylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate(Intermediate 20b, alternative procedure)

A solution of tert-butyl 2,3-dihydropyrrole-1-carboxylate (500 mg, 2.95mmol) and 3-trimethylsilylprop-2-ynal oxime (459.06 mg, 3.25 mmol) inMTBE (15 mL) was cooled to 0-5° C. while stirring. Sodium hypochlorite(2.806 mL, 5.91 mmol) was added dropwise keeping the reactiontemperature below 20° C. The reaction mixture was stirred at the sametemperature for 3 hours; afterwards, it was quenched with Na₂SO₃solution; the two phases were separated, the organic layer was washedwith water and brine, dried over Na₂SO₄, filtered and evaporated todryness in vacuo. The crude residue was purified by automated flashchromatography (Biotage SP1, cartridge type SNAP25) using a gradientfrom petroleum ether:EtOAc 95:5 to 7:3. Further purification byautomated flash chromatography (Isolera Biotage) with a gradientPetroleum Ether:EtOAc from 5:5 to 0:10 afforded 250 mg of the titleproduct. Yield: 27%.

tert-butyvl-3-(3-fluorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate(Intermediate 43a)

The title compound was synthesized using the method reported above forIntermediate 20c, but replacing 2-bromo-6-methylpyridyne with1-fluoro-3-iodo-benzene. After the usual work-up procedure the residuewas purified by automated flash chromatography (Isolera Biotage;gradient Petroleum Ether:EtOAc from 95:5 to 7:3) to give the titlecompound. Yield: 76%.

MS: [M+H]⁺=331.65

3-(3-fluorophenylethynyl)-4,5,6,6a-tetrahydropyrrolo[3,2-d]isoxazole(Intermediate 43b)

The title compound was synthesized using the method reported above forIntermediate 6e, but replacing Intermediate 6d with Intermediate 43a.After the usual work-up procedure the residue was used for the next stepwithout further purification. Yield: 95% (crude product).

MS: [M+H]⁺=231.54

3-(3-fluorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-morpholin-4-yl-methanone

To a solution of Intermediate 43b (50 mg, 0.21 mmol) in CH₂Cl₂ (6 mL)was added TEA (56 μL, 0.43 mmol) and then, dropwise,morpholine-4-carbonyl chloride (38.1 μL, 0.32 mmol). The reaction washeated at 50° C. for 4 h. The reaction mixture was then poured intowater, the organic layer was separated, dried over Na₂SO₄ and evaporatedto dryness in vacuo. The crude product was purified by flashchromatography (Isolera® Biotage) eluting with a gradient petroleumether:ethyl acetate 8:2 to 2:8 affording the title compound as a whitesolid (0.31 g, 41% yield).

MS: [M+H]⁺=344.54

¹H NMR (400 MHz, DMSO-d6) ppm 7.45-7.58 (m, 3H) 7.34-7.43 (m, 1H) 6.54(d, 1H) 4.10-4.19 (m, 1H) 3.51-3.69 (m, 5H) 3.30-3.38 (m, 2H) 3.19-3.28(m, 2H) 3.12 (td, 1H) 2.05-2.25 (m, 2H)

Example 443-(3-fluorophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(pyrrolidin-1-yl)methanone

The title compound was synthesized using the method reported above forExample 43, but replacing 4-pyrrolidine carbonyl chloride formorpholine-4-carbonyl chloride. After the usual work-up procedure theresidue was purified by flash chromatography (Isolera® Biotage) elutingwith a gradient petroleum ether:ethyl acetate 8:2 to 2:8 affording thetitle compound as a white solid (0.20 g, 28% yield).

MS: [M+H]⁺=328.54

¹H NMR (400 MHz, DMSO-d6) ppm 7.45-7.58 (m, 3H) 7.29-7.44 (m, 1H) 6.54(d, 1H) 4.19 (dd, 1H) 3.68 (dd, 1H) 3.35-3.46 (m, 2H) 3.23-3.28 (m, 2H)3.11 (td, 1H) 2.04-2.28 (m, 2H) 1.59-1.93 (m, 4H)

Example 453-phenylethynyl-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(morpholin-4-yl)methanonetert-butyl-3-(2-phenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate(Intermediate 45a)

The title compound was synthesized using the method reported above forIntermediate 20c, but replacing 2-bromo-6-methylpyridyne withiodobenzene. After the usual work-up procedure the residue was purifiedby means of automated flash chromatography (Isolera-Biotage; gradientPetroleum Ether:EtOAc from 95:5 to 7:3) to give the title compound.

Yield: 59%.

MS: [M+H]⁺=313.51

3-(2-phenylethynyl)-4,5,6,6a-tetrahydropyrrolo[3,2-d]isoxazole(Intermediate 45b)

The title compound was synthesized using the method reported above forIntermediate 6e, but replacing Intermediate 6d with Intermediate 45a.After the usual work-up procedure the residue was used for the next stepwithout further purification Yield: 98% (crude product).

MS: [M+H]⁺=213.54

3-phenylethynyl-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(morpholin-4-yl)methanone

The title compound was synthesized using the method reported above forExample 43, but replacing Intermediate 43b with Intermediate 45b. Afterthe usual work-up procedure the residue was purified by flashchromatography (Isolera® Biotage) eluting with a gradient petroleumether:ethyl acetate 8:2 to 2:8 affording the title compound as a whitesolid (0.23 g, 25% yield).

MS: [M+H]⁺=326.55

¹H NMR (400 MHz, DMSO-d6) ppm 7.59-7.66 (m, 2H) 7.43-7.56 (m, 3H) 6.53(d, 1H) 4.13 (dd, 1H) 3.52-3.69 (m, 5H) 3.33-3.41 (m, 2H) 3.19-3.28 (m,2H) 3.13 (td, 1H) 2.05-2.24 (m, 2H)

Example 463-(3-bromophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(pyrrolidin-1-yl)methanone

tert-Butyl-3-(3-bromophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazole-6-carboxylate(Intermediate 46a)

The title compound was synthesized using the method reported above forIntermediate 20c, but replacing 2-bromo-6-methylpyridyne with1-bromo-3-iodo-benzene. After the usual work-up procedure the residuewas purified by means of automated flash chromatography(Isolera-Biotage; gradient Petroleum Ether:EtOAc from 9:1 to 6:4) togive the title compound. Yield: 42%.

MS: [M+H]⁺=392.66

3-(3-bromophenylethynyl)-4,5,6,6a-tetrahydropyrrolo[3,2-d]isoxazole(Intermediate 46b)

The title compound was synthesized using the method reported above forIntermediate 6e, but replacing Intermediate 6d with Intermediate 46a.After the usual work-up procedure the residue was used for the next stepwithout further purification Yield: 89% (crude product).

MS: [M+H]⁺=292.78

3-(3-bromophenylethynyl)-3a,4,5,6a-tetrahydropyrrolo[3,2-d]isoxazol-6-yl-(pyrrolidin-1-yl)methanone

The title compound was synthesized using the method reported above forExample 43, but replacing Intermediate 43b with Intermediate 46b and4-pyrrolidine carbonyl chloride for morpholine-4-carbonyl chloride.After the usual work-up procedure the residue was purified by flashchromatography (Isolera® Biotage) eluting with a gradient petroleumether:ethyl acetate 8:2 to 2:8 affording the title compound as a whitesolid (0.20 g, 19% yield).

MS: [M+H]⁺=389.71

¹H NMR (400 MHz, DMSO-d6) ppm 7.87 (m, 1H), 7.72 (m, 1H), 7.64 (m, 1H),7.39-7.48 (m, 1H), 6.54 (d, 1H), 4.07-4.18 (m, 1H), 3.60-3.72 (m, 1H),3.34-3.44 (m, 2H), 3.31 (d, 2H), 3.11 (m, 1H), 2.03-2.30 (m, 2H),1.64-1.94 (m, 4H).

All references cited in this application are incorporated by referencein their entirety.

The invention claimed is:
 1. A compound of Formula I;

or an enantiomer, diastereomer, N-oxide, or a pharmaceuticallyacceptable salt thereof, wherein: R₁ is chosen from the group consistingof an alkyl group, an optionally substituted mono-, bi- or tricyclicC₁-C₁₃ heterocyclic group containing 1 to 5 heteroatoms selected from N,O, and S; an optionally substituted mono-, bi- or tricyclic C₆-C₁₄ arylgroup, an optionally substituted C₃-C₆ cycloalkyl group, or anoptionally substituted C₃-C₆ cycloalkenyl group; CO, CS, CH, CH2 and anSO2 group optionally substituted by one or more R₂ groups orsubstituents; R₂ is chosen from the group consisting of an optionallysubstituted mono- or bicyclic C₁-C₉ heterocyclic group containing from 1to 3 heteroatoms selected from nitrogen, oxygen and sulfur, anoptionally substituted mono-, bi- or tricyclic C₆-C₁₄ aryl group, or anoptionally substituted group chosen from alkyl, cycloalkyl, alkoxy,cycloalkyloxy, aryloxy, heteroaryloxy, alkylthio, amino, N-alkylamino,N,N-dialkylamino, and N-alkyl-N-alkoxyamino; R₃ is chosen from the groupconsisting of an optionally substituted alkyl group, an optionallysubstituted mono-, bi- or tricyclic C₁-C₁₃ heterocyclic group containing1 to 5 heteroatoms selected from N, O, and S; an optionally substitutedmono-, bi- or tricyclic C₆-C₁₄ aryl group, an optionally substitutedC₃-C₆ cycloalkyl group, and an optionally substituted C₃-C₆ cycloalkenylgroup; and n is 1-3.
 2. The compound according to claim 1, wherein theoptional substituents being independently selected from the groupconsisting of halogen atoms and C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxy,mercapto, nitro, cyano, oxo, halo(C₁-C₆)alkyl, halo(C₁-C₆)alkoxy, C₁-C₆alkylthio, C₁-C₆ alkylsulphonyl, C₁-C₆ alkylcarbonyl, sulphamoyl, C₁-C₆alkylsulphamoyl, di(C₁-C₆)alkylsulphamoyl, (C₁₋C₆)alkoxycarbonyl and(C₁-C₆)alkylcarbonyl(C₁-C₆)alkyl groups, and from groups of the formulae—NR*R*, —C(═O)—NR*R*, -A, —O-A, —C(═O)-A, —(CH2)q-A, —NR**-A,—C(═O)—NR**-A, —NR**C(═O)-A and —O—C(═O)-A wherein each R* independentlyrepresents a hydrogen atom or a C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆alkylcarbonyl, phenyl or benzyl group, R** represents a hydrogen atom ora C₁-C₆ alkyl group, q is an integer from 1 to 6 and A represents aphenyl group or a C₁-C₈ heterocyclic group containing from 1 to 3heteroatoms selected from N, O and S; a C₁-C₆ cycloalkyl group; eachgroup A being optionally substituted with from 1 to 3 groupsindependently selected from halo, hydroxy, cyano, nitro and C₁-C₆ alkyl,preferably wherein the optional substituents are independently selectedfrom the groups consisting of halogen atoms and C₁-C₆ alkyl groups. 3.The compound according to claim 1, wherein n=1.
 4. The compoundaccording to claim 1, wherein R₁ is a CO group.
 5. The compoundaccording to claim 1, wherein R₂ is an optionally substituted mono- orbicyclic C₁-C₉ heterocyclic group containing from 1 to 3 heteroatomsselected from nitrogen, oxygen and sulfur, or an optionally substitutedgroup chosen from cycloalkyl, alkoxy, cycloalkyloxy, aryloxy,heteroaryloxy, amino, N-alkylamino, N,N-dialkylamino, orN-alkyl-N-alkoxy.
 6. The compound according to claim 5, wherein R₂ hasthe formula:—OR₄ where R4 is chosen from the group consisting of a C₁-C₁₀ linear orbranched alkyl group, a C₁-C₁₀ cycloalkyl group and a C₁-C₁₀heterocyclic group containing at least one heteroatom selected from N orO.
 7. The compound according to claim 5 wherein R₂ is chosen from thegroup consisting of a saturated or unsaturated, optionally substituted,five or six membered homocyclic group and heterocyclic group containingat least one heteroatom selected from N or O.
 8. The compound accordingto claim 5 wherein R₂ has the formula:—NR₅R₆ where R5 is chosen from the group consisting of a C₁-C₁₀ linearor branched alkyl, alkoxy group and hydrogen; R6 is a C₁-C₁₀ linear orbranched alkyl or alkoxy group, R₅ and R₆ being the same or different;or wherein R₅ and R₆ together with the nitrogen atom form a five or sixmembered heterocyclic ring.
 9. The compound according to claim 1,wherein R₃ is chosen from the group consisting of an optionallysubstituted mono-, bi- or tricyclic C₆-C₁₄ aryl group, an optionallysubstituted, five or six membered heterocyclic group containing at leastone heteroatom selected from N or O, an optionally substituted C₃-C₆cycloalkyl group, and an optionally substituted C₃-C₆ cycloalkenylgroup.
 10. The compound according to claim 9, wherein R₃ is a phenyl orpyridyl group, said optional substituents being selected from a C₁-C₁₀alkyl group or a halide group.
 11. The compound according to claim 1,wherein R₁ represents an optionally substituted CO group, R₂ is absent,R₃ represents a phenyl, 3-bromophenyl, 3-chlorophenyl, 3-fluorophenyl,3-methylphenyl or a 6-methyl-2-pyridyl group and n is
 1. 12. A compoundaccording to claim 1 selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 13. A compound accordingto claim 1 selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 14. A pharmaceuticalcomposition comprising a compound according to claim 1.